Bioengineered lactobacillus probiotics and the uses thereof

ABSTRACT

In some illustrative embodiments, the present disclosure relates to a method for treating or preventing an inflammatory condition of a patient comprising the step of administering a therapeutically effective amount of Next Generation Bioengineered Probiotics (NGBP), together with one or more pharmaceutically acceptable carriers, diluents, and excipients, to the patient in need of relief from said inflammatory condition. In some other embodiments, the present application relates to an animal feed supplement for improving animal health and meat production compromising Next Generation Bioengineered Probiotics (NGBP). Yet in some other embodiments, the present invention relates to method for improving animal health and/or meat production comprising the step of adding an effective amount of Next Generation Bioengineered Probiotics (NGBP) to the feed of said animal.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/458,243, filed Jul. 1, 2019, which relates toand claims the benefit of U.S. provisional application 62/692,880, filedon Jul. 2, 2018. The contents of which are expressly incorporated hereinby reference in its entirety into the present disclosure.

STATEMENT OF SEQUENCE LISTING

A computer-readable form (CRF) of the Sequence Listing is submittedconcurrently with this application. The file, generated on Jun. 28,2019, is entitled Sequence_Listing_68291-02_ST25_txt. Applicant statesthat the content of the computer-readable form is the same and theinformation recorded in computer readable form is identical to thewritten sequence listing.

TECHNICAL FIELD

The present application relates to a method for treating or preventingan inflammatory condition of a patient comprising the step ofadministering a therapeutically effective amount of Next GenerationBioengineered Probiotics (NGBP), together with one or morepharmaceutically acceptable carriers, diluents, and excipients, to thepatient in need of relief from said inflammatory condition. In someother embodiments, the present invention relates to method for improvinganimal health and/or meat production comprising the step of adding aneffective amount of Next Generation Bioengineered Probiotics (NGBP) tothe feed of said animal.

BACKGROUND

This section introduces aspects that may help facilitate a betterunderstanding of the disclosure. Accordingly, these statements are to beread in this light and are not to be understood as admissions about whatis or is not prior art.

Listeria monocytogenes is an opportunistic human foodborne pathogenresponsible for severe systemic infection (listeriosis), and abortion,stillbirth and premature birth in pregnant women, mortality in newborns,the elderly and other immunocompromised individuals. L. monocytogenes iswell adapted to survive in the harsh environment of the gut (Sleator etal., 2009; Xayarath and Freitag, 2012). For the systemic spread, L.monocytogenes overcomes intestinal epithelial innate defense (Vance etal., 2009) and crosses the epithelial barrier. M cells overlying Peyer'spatches (Marco et al., 1997; Pron et al., 1998) and Internalin A(InlA)-mediated pathways are considered common events for epithelialbarrier crossing. InlA interacts with the host cell receptor E-cadherinfor intracellular spread (Lecuit et al., 2001); however, it is locatedat the basolateral side of the epithelial adherens junction (AJ) and isinaccessible to luminal L. monocytogenes. It is proposed that E-cadherinexposed during villous epithelial cell extrusion (Pentecost et al.,2006) and mucus exocytosis (Nikitas et al., 2011), can interact withListeria InlA. InlA/E-cadherin interaction is host species-specific. Inmouse E-cadherin, proline is substituted by glutamic acid at the aminoacid sequence position 16, thus InlA has low affinity for mouse or ratE-cadherin but has a strong interaction with the E-cadherin ofpermissive hosts, such as humans, gerbils and guinea pigs (Lecuit etal., 1999). Studies using transgenic mice expressing “humanized”E-cadherin (Disson et al., 2008) or murinized InlA (InlA^(m)) (BouGhanem et al., 2012; Wollert et al., 2007) have indicated that L.monocytogenes may use alternate routes to translocate across the gutmucosa. We recently showed that L. monocytogenes, uses Listeria adhesionprotein (LAP) to cross the intestinal epithelium by inducing epithelialbarrier dysfunction by activating NF-kB and MLCK, in the absence ofInlA, in epithelial cell and mouse models (Burkholder and Bhunia, 2010;Drolia et al., 2018). In mice, the bacterium is found in the epitheliallamina propria, mesenteric lymph nodes (MLN), blood, liver, spleen, andkidneys.

LAP (866 aa) is a housekeeping alcohol acetaldehyde dehydrogenase(Jagadeesan et al., 2010) in L. monocytogenes and displays moonlightingactivity (See below and Sequence Listing for details). It interacts withthe host cell Hsp60 (Wampler et al., 2004), a mammalian moonlightchaperone protein (Henderson et al., 2013), activates NF-kB leading tothe proinflammatory cytokines release, myosin light chain kinase (MLCK)upregulation and epithelial tight junction protein mislocalization(claudin-1, occludin and E-cadherin), leading to a leaky epithelialbarrier for bacterial passage (Drolia et al., 2018).

LAP protein sequence from Listeria monocytogenes (SEQ ID NO: 1): 1maikenaaqe vlevqkvidr ladngqkalk afesynqeqv dnivhamala gldqhmplak 61laveetgrgl yedkciknif ateyiwnnik nnktvgvine dvqtgvieia epvgvvagvt 121pvtnptsttl fkaiiaiktr npiifafhps aqrcssaaak vvydaaiaag apehciqwve 181kpsleatkql mnhdkvalvl atggagmvks aystgkpalg vgpgnvpayi dktakikrsv 241ndiilsksfd qgmicaseqa vivdkevake vkaemeankc yfvkgaefkk lesyvinpek 301gtlnpdvvgk spawianqag fkvpedtkil vaeikgvgdk yplsheklsp vlafieaanq 361aeafdrceem lvygglghsa vihstdkevq kafgirmkac riivnapsaq ggigdiyngf 421ipsltlgcgs ygknsvsqnv satnllnvkr iadrrnnmqw fklppkiffe kystqylqkm 481egvervfivt dpgmgsfkyv dvviehlkkr gndvayqvfa dvepdpsdvt vykgaelmkd 541fkpdtiialg ggsamdaakg mwlfyehpea sffglkqkfl dirkrtfkyp klggkakfva 601ipttsgtgse vtpfavitdk ennikyplad yeltpdvaiv daqyvttvpa hitadtgmdv 661lthaiesyvs vmasdytrgl siraielvfe nlresvltgd pdarekmhna salagmafan 721aflginhsla hkigpefhip hgranailmp hvirynalkp kkhalfprye sfradedyar 781isriigfpaa tteegvkslv deiiklgkdv gidmslkgqn vakkdldavv dtladrafmd 841qcttanpkqp lvselkeiyl eaykgv LAP protein sequence fromListeria innocua (SEQ ID NO: 2):  1 maikenaaqe vlevqkvidr ladngqkalkafesynqeqv dnivhamala gldqhmplak 61 laveetgrgl yedkciknif ateyiwnniknnktvgvine dtqtgvieia epvgvvagvt 121 pvtnptsttl fkaiiaiktr npiifafhpsaqrcsseaak vvydaavaag apehciqwve 181 kpsleatkql mnhdkvalvl atggagmvksaystgkpalg vgpgnvpayi dktakikrsv 241 ndiilsksfd qgmicaseqa vivdkevakevkaemeankc yfvkgaefkk lesyvinpek 301 gtlnpdvvgk spawianqag fkvpedtkilvaeikgvgdk yplsheklsp vlafieaatq 361 aeafdrceem lvygglghsa vihstdkevqkafgirmkac riivnapsaq ggigdiyngf 421 ipsltlgcgs ygknsvsqnv satnllnvkriadrrnnmqw fklppkiffe kystqylqkm 481 egvervfivt dpgmvqfkyv dvviehlkkrgndvayqvfa dvepdpsdvt vykgaelmkd 541 fkpdtiialg ggsamdaakg mwlfyehpeasffglkqkfl dirkrtfkyp klggkakfva 601 ipttsgtgse vtpfavitdk ennikypladyeltpdvaiv daqyvttvpa hitadtgmdv 661 lthaiesyvs vmasdytrgl siraielvfenlresvltgd pdarekmhna salagmafan 721 aflginhsla hkigpefhip hgranailmphvirynalkp kkhalfprye sfradedyar 781 isriigfpaa tteegvkslv deiiklgkdvgidmslkgqn vakkdldavv dtladrafmd 841 qcttanpkqp lvselkeiyl eaykgv

The gut mucosa represents the first site for the dynamic interaction ofthe enteric pathogens with the host (Finlay and Falkow, 1997).Therefore, averting this critical pathogen interaction step should helpprevent extra-intestinal dissemination of pathogens and the consequentpathology. Live probiotics bacteria such as lactobacilli andbifidobacteria are known to colonize and proliferate in the intestine toimprove intestinal microbial balance and protect the host from pathogens(Cross, 2002; Salminen et al., 2010). Among the different probioticbacteria used, Lactobacillus species are common because they are naturalinhabitants of the gut, modulate immune system (Amalaradjou and Bhunia,2012; Sanders et al., 2014), and enhance epithelial innate defense andrestore epithelial barrier function (Bron et al., 2017; Pagnini et al.,2010).

One of the major drawbacks of probiotics for prophylactic or therapeuticuse is that the antimicrobial effect is inconsistent and may be strainspecific (Hill et al., 2014) thus may have limited efficacy against atarget pathogen. Therefore, there are unmet needs in using probioticbacteria to prevent pathogen interactions with the host (Amalaradjou andBhunia, 2013; Focareta et al., 2006; Michon et al., 2016; Mohamadzadehet al., 2010).

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features, and advantages of the presentinvention will become more apparent when taken in conjunction with thefollowing description and drawings wherein identical reference numeralshave been used, where possible, to designate identical features that arecommon to the figures, and wherein:

FIGS. 1A-1D demonstrates that Listeria Adhesion Protein (LAP) fromListeria innocua restored adhesion and translocation ability of thelap-deficient L. monocytogenes (KB208) to enterocytes. FIG. 1A showsamino acid sequence comparison of LAP from L. monocytogenes (SEQ IDNO: 1) and L. innocua (SEQ ID NO: 2). FIG. 1B shows Western blotanalysis of protein preparation from cell wall showing expression of L.innocua LAP in L. monocytogenes KB208 (KB208LAP^(Lin)). FIG. 1C showsadhesion of KB208LAP^(Lin) to Caco-2 cells FIG. 1D shows translocationof KB208LAP^(Lin) across Caco-2 cells. Data represent three experimentsran in duplicate. *, P=0.05, **, P=0.001; ***, P=0.0001, ns=notsignificant.

FIGS. 2A-2F show bioengineered Lactobacillus Probiotic (BLP) expressingLAP (Listeria adhesion protein) of L. innocua reduced L. monocytogenesinfection in Caco-2 model. FIG. 2A shows the analysis of LAP expressionin bioengineered L. casei: Western blot showing LAP expression in cellwall fractions of L. casei expressing LAP of L. monocytogenes(LbcLAP^(Lm)), and L. innocua (LbcLAP^(Lin)). Purified recombinant LAPof L. monocytogenes (rLAP^(Lm)) was used as a positive control. FIG. 2Bshows translocation of bioengineered probiotics across Caco-2 cells.FIG. 2C shows inhibition of L. monocytogenes adhesion; and FIG. 2D showstransepithelial translocation of Listeria monocytogenes (Lm) on Caco-2cell line treated with L. casei (Lbc) and LbcLAP^(Lm), LbcLAP^(Lin), andL. casei carrying empty plasmid vector, pLP401T without any insert(LbcVeclap-). FIG. 2E shows increased co-aggregation of BLP (LbcLAP^(Lm)and LbcLAP^(Lin)) strains in co-incubated suspensions containing equalnumbers of BLP+Lm cells captured via Listeria-specific immunomagneticbeads (IMB). FIG. 2F depicts micrographs showing co-aggregated BLP cells(LbcLAP^(Lm) and LbcLAP^(Lin)) with IMB-captured Lm cells expressingGFP. Bars, 1 μm.

FIGS. 3A-3H show characterization of bioengineered probiotic strains.FIGS. 3A-3B show immunofluorescence staining and flow cytometry usinganti-LAP mAb-H7 to verify LAP expression on bioengineered probioticsbacteria. FIG. 3C depicts the absence of bacteriocin-like antimicrobialactivity in LbcWT and bioengineered strains (LbcLAP^(Lin); LbcLAP^(Lm))against L. monocytogenes lawn. Pediocin (a bacteriocin from Pediococcusacidilactici), and vancomycin were used as positive controls showing azone of inhibition. FIGS. 3D-3F show survival of bioengineered L. casei(LbcLAP^(Lin); LbcLAP^(Lm)) and LbcWT in simulated gastric fluid (SGF)(3D), simulated intestinal fluid I (SGF-I) (3E), and simulatedintestinal fluid II (SGF-II) (3F). FIG. 3G shows the light microscopicphotographs showing the live and dead stained bioengineered LbcLAP^(Lin)strain using cFDA-SE (carboxyfluorescein diacetate succinimidyl ester)and PI (propidium iodide) after exposure to simulated gastric fluid(SGF) and simulated intestinal fluid (SIF) for 2.5 h. FIG. 3H confirmsLAP expression in bioengineered probiotics (LbcLAP^(Lin); LbcLAP^(Lm)),but absent in LbcWT when grown SIF-I.

FIGS. 4A-4K demonstrate that bioengineered Lactobacillus casei reducedL. monocytogenes infection in a mouse (A/J) model. FIG. 4A is aschematics showing animal experiment protocol: mice (female A/J mice, 6weeks old) were fed probiotics for 10 days, and then challenged with L.monocytogenes F4244 (8.8×10⁸ cfu/animal). FIG. 4B Mice body weightanalysis over 12-days period during probiotic feeding and challenge withL. monocytogenes (Lm) at time points 0, 5, 10, and 12 days. FIGS. 4C-4Dshow analysis of bioengineered Lbc colonization in the mouse gut: (4C)Total lactic acid bacterial counts in animals that were fed withdifferent bioengineered Lbc or controls on MRS agar plate and vancomycinresistant LbcWT, LbcLAP^(Lin); LbcLAP^(Lm) (4D) in the intestine andfeces during 10 days of feeding. Wild type and bioengineered probioticcounts in the intestine and fecal samples of mice from day 13. MRScontaining vancomycin (300 μg/ml) was used to isolate LbcWT (n=15 mice)and MRS containing erythromycin (2 μg/ml) was used to enumeratebioengineered probiotics, LbcLAP^(Lin) (n=15) and LbcLAP^(Lm) (n=15). Asexpected, no antibiotic resistant probiotics were detected from controlanimals or control animals that received L. monocytogenes (Lm) only.

FIGS. 4E-4K depict mice experiments showing bioengineered probioticmediated prevention of L. monocytogenes infection. L. monocytogenescounts in probiotic fed mice in (4E) liver, (4F) spleen, (4G) MLN, (4H)kidney, (4I) blood, (4J) intestine, and (4K) feces after 24 h or 48 hpost infection. (n=6-10 mice). Each animal was represented by a dot inthe plot (n=3-10 mice per group). Horizontal dotted lines indicatedetection limit of the assay. Treatments were, wild type L. casei(LbcWT), bioengineered L. casei expressing LAP of L. monocytogenes(LbcLAP^(Lm)) and L. innocua (LbcLAP^(Lin)). No background Listeria wasdetected from mice that received only the probiotics (LbcWT, LbcLAPLm,LbcLAPLin) or no probiotics at all. Data were analyzed by Man Whitneytest using GraphPad Prism 6. (*, P=0.05, **, P=0.001; ***, P=0.0001,ns=not significant).

FIG. 5 shows visual examination of health status of mice afterchallenged with L. monocytogenes. The animals in the left panels(a,c,e,g) were not challenged (control), while the right panels(b,d,f,h) were challenged with L. monocytogenes F4244. The clinicalonset of listeriosis in (b) No Lbc+Lm and (d) LbcWT+Lm was evident.LbcLAP^(Lin)+Lm mice appeared healthy.

FIGS. 6A-6D demonstrate epithelial permeability assessment afterprobiotic exposure. FIGS. 6A-6B show epithelial permeability in Caco-2cell monolayers in transwell insert using 4 kDa FITC-Dextran (FD4) (a)movement from apical to basolateral side and Transepithelial electricalresistance (TEER) (b) after treatment with Control, L. monocytogenes(Lm), No Lbc+Lm, LbcWT, LbcWT+Lm, LbcLAP^(Lm), LbcLAP^(Lm)+Lm,LbcLAP^(Lin), LbcLAP^(Lin)+Lm. FIGS. 6A-6B show intestinal epithelialpermeability assessment by measuring FD4 levels in serum (6C) and urine(6D) in probiotic fed mice from FIGS. 4A-4K. Treatments were, control,No Lbc+Lm, LbcWT, LbcWT+Lm, LbcLAP^(Lm), LbcLAP^(Lm)+Lm, LbcLAP^(Lin),LbcLAP^(Lm)+Lm. Bioengineered probiotics significantly reduced the FD4translocation compared to the LbcWT or Lm alone. (***, P<0.0001; *,P<0.05, ns, not significant).

FIGS. 7A-7C show cellular junctional protein distribution analysis inCaco-2 and ileal tissue of mice. FIG. 7A shows Western blot showingtight junction (ZO-1, occludin, claudin-1) and adherence junctionprotein (E-cadherin) levels in cells after treatment with L.monocytogenes or probiotic bacteria followed by L. monocytogeneschallenge. Confirmation of cell-junction protein mislocalization byconfocal immunofluorescence microscopy in Caco-2 cells (7B) and in mouseileal tissue section (7C). White arrows (presence) and yellow (absenceor mislocalization) pointing to the cell junction proteins.

FIGS. 8A-8E show histopathological scoring of ileal tissues ofbioengineered probiotic-fed mice. (8A) H&E stained sections, (8B)Histology score, (8C & 8D) Increased goblet cell counts in bioengineeredprobiotic-fed mice ileal tissues. (8E) Immunostaining of tissue sectionsfor Hsp60 expression, (8F) analysis of transcripts of hspdl (hsp60) inileal tissues in probiotic fed mice. Treatments were, control, NoLbc+Lm, LbcWT, LbcWT+Lm, LbcLAP^(Lm), LbcLAP^(Lm)+Lm, LbcLAP^(Lin),LbcLAP^(Lin)+Lm. (8E & 8F) In the presence of L. monocytogenes,epithelial cells expressed a high level of Hsp60 irrespective ofprobiotic treatment. Data were analyzed by one way ANOVA, and Tukey'sgrouping was used to determine statistical significance at ***,P<0.0001; *, P<0.05, ns, not significant).

FIGS. 9A-91 show immunomodulatory and anti-inflammatory effects ofprobiotics in RAW macrophage cell line and in mice. (9A & 9B)Attenuation of NF-kB expression in the luciferase reporter RAW cell line(a) and mouse ileum (b). Confocal imaging shows attenuation of NF-kBactivity by probiotics compared to Lm WT (b). (9C & 9D) TNF-α and IL-6expression in mice ilea after probiotic exposure. (9E-9G) Flow cytometryanalysis showing spleen CD4 cell levels were unaffected while CD8 andCD11c cell levels were increased after challenged with probioticsfollowed by Lm challenge. (9H) Spleen cytology score and (9I) lightmicroscopic imaging showed an increased inflammatory response incharacterized by infiltration of neutrophils, macrophages, andlymphocytes, and blinded cytology score to probiotic pre-exposurefollowed by L. monocytogenes infection. Treatments were, control, NoLbc+Lm, LbcWT, LbcWT+Lm, LbcLAP^(Lm), LbcLAP^(Lm)+Lm, LbcLAP^(Lin),LbcLAP^(Lin)+Lm. (n=3-10 mice/group). Two-way ANOVA and one-tailedT-tests of individual probiotic treatment pairs were used todemonstrates a significance at P<0.05.

FIGS. 10A-10D. Immunomodulatory action of probiotics expressing the LAPprotein: Ileum harvested from A/J mice supplied with or withoutprobiotics for ten days followed by 48 h-post infection with L.monocytogenes was immunostained for CD3 (10A), CD8α (10B), Fox-P3 (10C),and cleaved caspase-3 (10D, CC-3; marker of apoptosis. Graphs show CD3⁺,CD8α⁺, CC-3⁺ and CD4⁺ Fox-P3+, cells from 25 villi/mouse. Each pointrepresents an individual mouse. Mice fed with probiotics expressing theLAP protein (LbcLAP^(Lm) or LbcLAP^(Lin)) prior to L. monocytogenesinfection show significantly reduced CD3⁺, CD8⁺ (arrows), CC-3+ cellsbut significantly increased CD4⁺ Foxp3⁺ cells.

FIG. 11 shows survival of mice (A/J strain) supplied with or withoutprobiotics for ten days in drinking water followed by oral infectionwith L. monocytogens (n=at least 10 per group). Mice fed withLactobacillus casei (probiotics) expressing the LAP protein(LbcLAP^(Lin)) showed significantly (**P<0.01, Kaplan-Meier log-ranktest) higher survival compared to that of LbcWT.

FIG. 12. Comparison of the amino acid sequence of LAP (Listeria adhesionprotein) from different strains of Lm and Lin obtained from the NCBIdatabase.

FIG. 13. Confirmation of Bioengineered Lactobacillus probiotic (BLP)strains expressing LAP from Listeria innocua (LbcLAP^(Lin)) or L.monocytogenes (LbcLAP^(Lm)) by Western blotting (left panel) andconfocal imaging (right panel; arrows)

FIG. 14. BLP strains expressing LAP from Listeria innocua (LbcLAP^(Lin))or L. monocytogenes (LbcLAP^(Lm)) prevent Lm interaction in vitro in aCaco-2 cell culture model. Increased inhibition of Lm adhesion (left,n=6), invasion (middle, n=6) and translocation (right, n=6) by the BLPstrains after 24 h exposure to Caco-2 cells.

FIG. 15. Schematics showing mouse experiment protocol.

FIG. 16. BLP strains expressing LAP from Listeria innocua (LbcLAP^(Lin))or Lm (LbcLAP^(Lin)) prevent Lm induced weight loss in mice. Normalizedmouse body weight (mean±SD, n=5) on day 0, 5, 10, and 12.

FIG. 17. BLP strains expressing LAP from Listeria innocua (LbcLAP^(Lin))or Lm (LbcLAP^(Lin)) show increased intestinal colonization. IncreasedBLP counts in the intestinal content of mice (n=5) on days 10, 11, and12.

FIG. 18. BLP strains expressing LAP from Listeria innocua (LbcLAP^(Lin))or Lm (LbcLAP^(Lin)) prevent Lm intestinal infection in a mouse model.Reduced Lm burdens in the intracellular location in the ileum (left,n=6), cecum (middle, n=6), and colon (right, n=6).

FIG. 19. BLP strains expressing LAP from Listeria innocua (LbcLAP^(Lin))or Lm (LbcLAP^(Lm)) prevent Lm systemic infection in a mouse model.Reduced Lm burdens in the liver (left), spleen (right) (left and right,n=5 at 24 h post infection (hpi); and n=17, 14, 13, 14 at 48 hpi), MLN(center, n=11, 9, 8, 9, for each group, respectively),

FIG. 20. BLP strains expressing LAP from Listeria innocua (LbcLAP^(Lin))or Lm (LbcLAP^(Lm)) prevent Lm systemic infection in a mouse model.Reduced Lm burdens in the blood (right, n=5 at 24 hpi; and n=11, 8, 8,8, at 48 hpi for each group, respectively) and kidney (left, n=6, 3, 3,3, for each group, respectively).

FIG. 21. BLP strains expressing LAP from Listeria innocua (LbcLAP^(Lin))or Lm (LbcLAP^(Lm)) prevent Lm intestinal penetration in a mouse model.Micrographs of ileal (left) villi immunostained for ZO-1 (brown) and Lm(red, arrows) and counterstained for nucleus (blue) at 48 hpi. Bars, 10μm. The boxed areas were enlarged. Bars, 1 μm. Lm counts (mean±SEM) inileal lamina propria (LP; right panels). Dots represent an average of 25villi from a single mouse, four mice/group, n=100 villi. Lm is observedin the LP (arrows) in naive or LbcWT-treated mice but confined in thelumen (arrows) in BLP-treated mice (Lbc LAP^(Lin) and Lbc LAP^(Lm)).

FIG. 22. BLP (LbcLAP^(Lin)) prevents lethal L. monocytogenes infectionin mice. Increased survival of BLP-treated mice (LbcLAP^(Lin)) at LD₅₀dose. *p<0.05 Kaplan-Meier log-rank test.

FIGS. 23A-23B. BLP colonization and persistence in the intestine limitsLm translocation despite discontinuous administration. Schematics (FIG.23A) showing mouse experiment protocol: Mice were treated with L. casei(LbcWT) or BLP (LbcLAP^(Lin)) strain supplied in drinking waterreplenished daily (4-8×10⁹ CFU/ml) for 10 days (0-9 days) and thenchallenged with Lm F4244 (˜5×10⁸ CFU/animal) on day 10, 15 and 20. (FIG.23B) BLP-mediated reduced Lm burdens at 48 hpi in the intracellularlocation in the ileum (left), cecum (center), and colon (right) (allbottom panels, gentamicin resistant CFU), in mice on day 12

FIG. 24. BLP displays increased co-aggregation with Lm. Increasedco-aggregation of BLP (left panel, LbcLAP^(Lin,) and LbcLAP^(Lm))strains in co-incubated suspensions containing equal numbers of BLP+Lmcells (n=4) captured via Listeria-specific immunomagnetic beads (IMB)but not with BLP+L. innocua (Lin, n=6) cells or L. casei (alone) (n=10).Micrographs (right panel) showing co-aggregated BLP cells (arrows) withIMB-captured Lm cells expressing GFP (b). Bars, 1 μm.

FIG. 25. BLP (LbcLAP^(Lin) and LbcLAP^(Lm)) forms increased biofilm onplastics. Increased biofilm formation (Abs 595 nm, mean±SEM) of BLPstrains as measured by crystal violet staining in monoculture andco-culture with grown in microtiter plates. Images (top panel) showcrystal violet stained biofilms of representative wells for eachtreatment. Data represent three independent experiments obtained fromn=6 independent microtiter plate wells.

FIG. 26. BLP (LbcLAP^(Lin) and LbcLAP^(Lm)) forms increased biofilm onmouse colonic villi (right two panels)

FIG. 27. BLP prevents Lm from causing intestinal barrier damage in amouse model. Representative H&E-stained micrographs (bars, 25 μm) (left)and the histological score (right, each point represents an individualmouse) of ileal tissue sections from control (mock-treated) uninfectednaïve mice or L. casei-treated (10 days, LbcWT or BLP) pre orpost-Lm-challenge at 48 hpi (n=9, 7, 7, 9, 9, 9, 10, 11 mice for eachgroup, respectively). Arrows point to the loss of villous epithelialcells and increased polymorphonuclear and mononuclear cells infiltratingthe base of the villous lamina propria in naïve (naive+Lm) and LbcWT-treated mice (LbcWT+Lm) at 48 hpi.

FIG. 28. BLP prevents Lm from causing intestinal barrier loss bymaintaining mucus-producing goblet cells in mice. Representativeimmunohistochemical micrographs of the ileum stained for Muc2 (left,brown), nuclei (blue) from control (mock-treated) uninfected naïve miceor L. casei-treated (10 days, LbcWT or BLP) pre or post-Lm challenge at48 hpi. Bars, 10 μm. Quantification of Muc2 (right)-positive cells, eachpoint represents an individual mouse, 4 mice per group, n=100 villi.Arrows point to increased numbers of Muc2 (left) in BLP treated mice(pre- or post-Lm challenge).

FIG. 29: BLP blocks Lm from causing disturbance of intestinal epithelialcell-cell junctional integrity. FITC conjugated 4 kDa dextran (FD4) gutpermeability of control (mock-treated) naïve uninfected mice or L.casei-treated (10 days, LbcWT or BLP) pre- or post-Lm challenge (48 hpi)in serum (c) and urine (d). Each point represents an individual mouse.Data represent mean±SEM of n=3 mice for all groups except Lm group, n=5mice.

FIG. 30. BLP blocks Lm from causing disturbance of intestinal epithelialcell-cell junctional integrity. Immunofluorescence micrographs of theileal tissues showing increased expression of MLCK and P-MLC (green;arrows) and mislocalization (intracellular puncta) of claudin-1 (green;arrows), occludin, and E-cadherin (red; arrows) in naïve orLbcWT-treated (10 days) but baseline expression of MLCK and P-MLC andintact localization of occludin, claudin-1 and E-cadherin in BLP-treatedmice (10 days) at 48 hpi, relative to uninfected naïve mice. Nuclei;DAPI, blue. Images are representative of five different fields from n=3mice per treatment. Bars, 10 μm. LP, Lamina Propria.

FIG. 31: BLP prevents Lm-induced NF-κB (p65) activation and modulatescytokine production and immune cells to maintain intestinal immunehomeostasis. Immunofluorescence micrographs of the ileal tissues showingdecreased nuclear localization of p65 (green) in BLP-treated mice (10days) at 48 hpi. Nuclei; DAPI, blue. Arrows indicate the nuclearlocalization of p65 in IEC of naïve or LbcWT-treated (10 days) mice at48 hpi. The right panels show the quantified results (mean±SEM) of p65nuclear positive IEC. Each point represents an average of 15 villi froma single mouse, 4 mice per group, n=60 villi. Bars, 10 μm.

FIG. 32: BLP modulates the cytokine TNFα to maintain intestinal immunehomeostasis. ELISA showing decreased TNFα (n=6 mice for all groupsexcept LbcWT and LbcLAP^(Lin)+Lm group; n=5 and 4 mice, respectively),in the ileal tissues of BLP-treated (10 days) mice, relative to naïve orLbcWT-treated (10 days) at 48 hpi.

FIG. 33: BLP modulates the cytokine IL-6 to maintain intestinal immunehomeostasis. ELISA showing decreased IL-6 (n=6 mice for all groupsexcept LbcWT and LbcLAP^(Lin)+Lm group; n=5 and 4 mice, respectively),in the ileal tissues of BLP-treated (10 days) mice, relative to naïve orLbcWT-treated (10 days) at 48 hpi

FIG. 34: BLP modulates the cytokine IFNγ to maintain intestinal immunehomeostasis. ELISA showing increased IFNγ (n=4 mice for all groups), inthe ileal tissues of BLP-treated (10 days) mice, relative to naïve orLbcWT-treated (10 days) at 48 hpi

FIG. 35: BLP modulates the cytokine IL-10 to maintain intestinal immunehomeostasis. Graph showing increased IL-10⁺ cells quantified (mean±SEM)from immunostained ileal tissues (of BLP-treated (10 days) mice pre- orpost-Lm challenge at 48 hpi. Each point represents an average of 25villi from a single mouse, 4 mice per group, n=100 villi.

FIG. 36: BLP modulates the cytokine TGFβ⁺ to maintain intestinal immunehomeostasis. Graph showing increased TGFβ⁺ cells quantified (mean±SEM)from immunostained ileal tissues of BLP-treated (10 days) mice pre- orpost-Lm challenge at 48 hpi. Each point represents an average of 25villi from a single mouse, 4 mice per group, n=100 villi.

FIG. 37: BLP modulates CD4⁺ T-cell populations for immunomodulation.Representative immunohistochemical micrographs of ileal tissues showingincreased CD4⁺ cells (left, brown, arrows), in BLP-treated mice (10days) mice pre or post-Lm challenge at 48 hpi, relative to naïve orLbcWT-treated (10 days). Bars, 10 μm. Quantification of CD4⁺ cells(right), Each point represents an average (mean±SEM) of 25 villi from asingle mouse, 4 mice per group, n=100 villi.

FIG. 38: BLP modulates FOXP3⁺ T-regulatory cells populations forimmunomodulation. Representative immunohistochemical micrographs ofileal tissues showing increased FOXP3⁺ T-regulatory cells (left, brown,arrows), in BLP-treated mice (10 days) mice pre or post-Lm challenge at48 hpi, relative to naïve or LbcWT-treated (10 days). Bars, 10 μm.Quantification of FOXP3⁺ T-regulatory cells (right), Each pointrepresents an average (mean±SEM) of 25 villi from a single mouse, 4 miceper group, n=100 villi.

FIG. 39: BLP modulates CD11c⁺ dendritic cells populations forimmunomodulation. Representative immunohistochemical micrographs ofileal tissues showing increased CD11c⁺ dendritic cells (left, brown,arrows), in BLP-treated mice (10 days) mice pre or post-Lm challenge at48 hpi, relative to naïve or LbcWT-treated (10 days). Bars, 10 μm.Quantification of CD11c⁺ cells (right), Each point represents an average(mean±SEM) of 25 villi from a single mouse, 4 mice per group, n=100villi.

FIG. 40: BLP modulates natural-killer NKp46⁺ immune cell populations forimmunomodulation. Representative immunohistochemical micrographs ofileal tissues showing increased NKp46⁺ cells (left, brown, arrows) inBLP-treated mice (10 days) mice pre or post-Lm challenge at 48 hpi,relative to naïve or LbcWT-treated (10 days). Bars, 10 μm.Quantification of NKp46⁺ cells (right), Each point represents an average(mean±SEM) of 25 villi from a single mouse, 4 mice per group, n=100villi.

FIG. 41. Schematics showing the mechanism of BLP-mediated protectionagainst listeriosis. The BLP prevents Lm Infection by three mechanisms(i) Competitive exclusion, (ii) improved intestinal barrier function,and (iii) contact-dependent immunomodulation.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to the embodimentsillustrated in the drawings, and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of this disclosure is thereby intended.

In the present disclosure the term “about” can allow for a degree ofvariability in a value or range, for example, within 10%, within 5%, orwithin 1% of a stated value or of a stated limit of a range.

In the present disclosure the term “substantially” can allow for adegree of variability in a value or range, for example, within 70%,within 80%, within 90%, within 95%, or within 99% of a stated value orof a stated limit of a range.

The term “patient” includes human and non-human animals such ascompanion animals (dogs and cats and the like) and livestock animals.Livestock animals are animals raised for food production. The patient tobe treated is preferably a mammal, in particular a human being.

The term “pharmaceutically acceptable carrier” is art-recognized andrefers to a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting anysubject composition or component thereof. Each carrier must be“acceptable” in the sense of being compatible with the subjectcomposition and its components and not injurious to the patient. Someexamples of materials which may serve as pharmaceutically acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

As used herein, the term “administering” includes all means ofintroducing the compounds and compositions described herein to thepatient, including, but are not limited to, oral (po), intravenous (iv),intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal,ocular, sublingual, vaginal, rectal, and the like. The compounds andcompositions described herein may be administered in unit dosage formsand/or formulations containing conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles.

It is to be understood that the total daily usage of the compounds andcompositions described herein may be decided by the attending physicianwithin the scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular patient will depend upon avariety of factors, including the disorder being treated and theseverity of the disorder; activity of the specific compound employed;the specific composition employed; the age, body weight, general health,gender, and diet of the patient: the time of administration, and rate ofexcretion of the specific compound employed, the duration of thetreatment, the drugs used in combination or coincidentally with thespecific compound employed; and like factors well known to theresearcher, veterinarian, medical doctor or other clinician of ordinaryskill.

Depending upon the route of administration, a wide range of permissibledosages are contemplated herein, including doses falling in the rangefrom about 1 μg/kg to about 1 g/kg. The dosage may be single or divided,and may be administered according to a wide variety of dosing protocols,including q.d., b.i.d., t.i.d., or even every other day, once a week,once a month, and the like. In each case the therapeutically effectiveamount described herein corresponds to the instance of administration,or alternatively to the total daily, weekly, or monthly dose.

As used herein, the term “therapeutically effective amount” refers tothat amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinicians, which includes alleviation of the symptoms of thedisease or disorder being treated. In one aspect, the therapeuticallyeffective amount is that which may treat or alleviate the disease orsymptoms of the disease at a reasonable benefit/risk ratio applicable toany medical treatment.

As used herein, the term “therapeutically effective amount” refers tothe amount to be administered to a patient, and may be based on bodysurface area, patient weight, and/or patient condition. In addition, itis appreciated that there is an interrelationship of dosages determinedfor humans and those dosages determined for animals, including testanimals (illustratively based on milligrams per meter squared of bodysurface) as described by Freireich, E. J., et al., Cancer Chemother.Rep. 1966, 50 (4), 219, the disclosure of which is incorporated hereinby reference. Body surface area may be approximately determined frompatient height and weight (see, e.g., Scientific Tables, GeigyPharmaceuticals, Ardley, N.Y., pages 537-538 (1970)). It is appreciatedthat effective doses may also vary depending on the route ofadministration, optional excipient usage, and the possibility ofco-usage of the compound with other conventional and non-conventionaltherapeutic treatments, including other anti-tumor agents, radiationtherapy, and the like.

As used herein, a patient may be an animal or a human being.

Probiotic: The International Scientific Association of Probiotics andPrebiotics (ISAPP) in 2014 defined probiotics as “live microorganismsthat, when administered in adequate amounts, confer a health benefit onthe host” (Hill et al. Nat Rev Gastroenterol Hepatol 11.8 (2014):506-514).

Next Generation Probiotics (NGPs): Conform to the normal definition of aprobiotic, when administered in adequate amounts, confer a healthbenefit on the host and is applicable to the prevention, treatment, orcure of a disease or condition of human beings (O'Tolle et al. Naturemicrobiology 2.5 (2017): 17057; Langella et al. Frontiers inMicrobiology 10 (2019): 1047).

Next Generation Bioengineered Probiotics (NGBPs): Conform to the normaldefinition of NGPs, but are genetically modified probiotic strains toexclusively target a specific pathogen, toxin or disease conditions andcan be used for a therapeutic purpose (Amalaradjou et al Bioengineered4.6 (2013): 379-387; Hill et al. Nat Rev Gastroenterol Hepatol 11.8(2014): 506-514).

In some illustrative embodiments, the present invention relates to amethod for improving animal health and/or meat production comprising thestep of adding an effective amount of Next Generation BioengineeredProbiotics (NGBP) to the feed of said animal.

In some illustrative embodiments, the present invention relates to amethod for improving animal health and/or meat production comprising thestep of adding an effective amount of Next Generation BioengineeredProbiotics (NGBP) to the feed of said animal as disclosed herein,wherein said animal is selected from the group consisting of pig, sheep,goat, chicken, turkey, cat, dog, and cattle.

In some illustrative embodiments, the present invention relates to amethod for improving animal health and/or meat production comprising thestep of adding an effective amount of Next Generation BioengineeredProbiotics (NGBP) to the feed of said animal as disclosed herein,wherein said NGBP is a reengineered bacteria expressing Listeriaadhesion protein (LAP).

In some illustrative embodiments, the present invention relates to amethod for improving animal health and/or meat production comprising thestep of adding an effective amount of Next Generation BioengineeredProbiotics (NGBP) to the feed of said animal as disclosed herein,wherein said NGBP is a lyophilized product.

In some other illustrative embodiments, the present invention relates toan animal feed supplement for improving animal health and meatproduction compromising Next Generation Bioengineered Probiotics (NGBP).

In some illustrative embodiments, the present invention relates to ananimal feed supplement for improving animal health and meat productioncompromising Next Generation Bioengineered Probiotics (NGBP) asdisclosed herein, wherein said NGBP is a reengineered bacteriaexpressing Listeria adhesion protein (LAP).

In some illustrative embodiments, the present invention relates to ananimal feed supplement for improving animal health and meat productioncompromising Next Generation Bioengineered Probiotics (NGBP) asdisclosed herein, wherein said animal feed supplement is a lyophilizedproduct.

In some illustrative embodiments, the present invention relates to ananimal feed supplement for improving animal health and meat productioncompromising Next Generation Bioengineered Probiotics (NGBP) asdisclosed herein, wherein said animal is selected from the groupconsisting of pig, sheep, goat, chicken, turkey, cat, dog, and cattle.

Yet in some other embodiments, the present invention relates to a methodto reduce or eliminate antibiotics used in an animal feed for improvinganimal health and meat production comprising the step of adding aneffective amount of Next Generation Bioengineered Probiotics (NGBP) tothe feed.

In some other embodiments, the present invention relates to a method toreduce or eliminate antibiotics used in an animal feed for improvinganimal health and meat production comprising the step of adding aneffective amount of Next Generation Bioengineered Probiotics (NGBP) tothe feed as disclosed herein, wherein said animal is selected from thegroup consisting of pig, sheep, goat, chicken, turkey, cat, dog, andcattle.

In some other embodiments, the present invention relates to a method toreduce or eliminate antibiotics used in an animal feed for improvinganimal health and meat production comprising the step of adding aneffective amount of Next Generation Bioengineered Probiotics (NGBP) tothe feed as disclosed herein, wherein said NGBP is a reengineeredbacteria expressing Listeria adhesion protein (LAP).

In some other embodiments, the present invention relates to a method fortreating or preventing an inflammatory condition of a patient comprisingthe step of administering a therapeutically effective amount of NextGeneration Bioengineered Probiotics (NGBP), together with one or morepharmaceutically acceptable carriers, diluents, and excipients, to thepatient in need of relief from said inflammatory condition.

In some other embodiments, the present invention relates to a method fortreating or preventing an inflammatory condition of a patient comprisingthe step of administering a therapeutically effective amount of NextGeneration Bioengineered Probiotics (NGBP), together with one or morepharmaceutically acceptable carriers, diluents, and excipients, to thepatient in need of relief from said inflammatory condition as disclosedherein, wherein said inflammatory condition comprises Crohn's disease(CD), inflammatory Bowel Disease (IBD), and ulcerative colitis (US),wherein intestinal mucosal cells express a high level of Hsp60.

In some other embodiments, the present invention relates to a method fortreating or preventing an inflammatory condition of a patient comprisingthe step of administering a therapeutically effective amount of NextGeneration Bioengineered Probiotics (NGBP), together with one or morepharmaceutically acceptable carriers, diluents, and excipients, to thepatient in need of relief from said inflammatory condition as disclosedherein, wherein said NGBP is a reengineered bacteria expressing Listeriaadhesion protein (LAP).

In some other embodiments, the present invention relates to a method fortreating or preventing an inflammatory condition of a patient comprisingthe step of administering a therapeutically effective amount of NextGeneration Bioengineered Probiotics (NGBP), together with one or morepharmaceutically acceptable carriers, diluents, and excipients, to thepatient in need of relief from said inflammatory condition as disclosedherein, wherein NGBP is administered orally.

Here, we investigated whether a probiotic bacterium expressing LAP cancompetitively exclude pathogen interaction on the host epithelial cell,thereby preventing listeriosis in a high-risk population in thebackground of the probiotic's natural beneficial attributes. In aprevious study, as a proof of concept, we showed that LAP of L.monocytogenes expressed on Lactobacillus paracasei was able to reduce L.monocytogenes interaction with the enterocyte-like Caco-2 cell model,however, its effectiveness in an animal model and the host response areunknown. Here, we investigated if the LAP especially from anon-pathogenic Listeria (L. innocua), can be used on a more commonlyused probiotic strain, Lactobacillus casei to competitively excludepathogen interaction in a mouse model. Here, we show that Lactobacilluscasei expressing LAP from a nonpathogenic bacterium, L. innocua,supplied to mice (A/J) in drinking water for 10 days, and subsequentlychallenged with L. monocytogenes was able to protect mice fromlisteriosis. This probiotic also significantly reduced L. monocytogenesburden in the extra-intestinal tissues, modulated proinflammatorycytokines levels, dampened NF-kB activity, and improved epithelialinnate defense and barrier function to protect mice from the infection.

Potential benefits of “Next Generation Bioengineered Probiotics (NGBP)”expressing Listeria adhesion protein (LAP) from a nonpathogenic Listeria(L. innocua) that binds to a mammalian cell receptor, Hsp60 on humanhealth:

-   -   Probiotics, in general, have positive effects on the gut via        their expression of antimicrobial agents, their colonization of        niches that might otherwise be occupied by pathogenic bacteria,        modulating cytokine levels, and their effects on the gut immune        system. Overall, these effects are anti-inflammatory.    -   A key ‘receptor’ for LAP is Hsp60, which is involved in both        chaperoning and immune system function. At low levels, Hsp60 is        anti-inflammatory, but at higher concentrations, it can take on        pro-inflammatory roles.    -   In many chronic inflammatory disease conditions such as Crohn's        Disease (CD), Inflammatory Bowel Disease (IBD), and Ulcerative        Colitis (UC), intestinal mucosal cells express a high level of        Hsp60.    -   Inflammatory disease disrupts gut barrier function thus gut        becomes leaky allowing luminal microbes and endotoxins to enter        blood circulation. NGBP through its interaction with Hsp60 can        maintain epithelial barrier integrity thus prevent endotoxin        from crossing gut epithelial barrier—thus maintain a gut health.    -   Targeting innate or exogenous microbe-induced Hsp60 in mucosal        cells with LAP-expressing NGBP may usefully repress inflammatory        processes in the gut, as well as some types of infectious        processes.        -   LAP-expressing NGBP localization and persistence in the gut            is also enhanced because of their interaction with mucosal            Hsp60.    -   LAP-expressing NGBP also prevents Listeria monocytogenes        colonization and systemic spread and provides protection against        severe disease as confirmed in a mouse model.    -   The LAP-expressing NGBP, by virtue of its Hsp60 targeting,        uniquely addresses stabilization of the gut to inflammatory and        microbiological challenges, as demonstrated in mouse studies.

On the other hand, meat animals such as swine encounters variousstressful situations throughout their life. Early in life, weaning, foodand water deprivation during transportation and heat are commonstressors. Stressors can reduce feed intake, exert inflammatoryresponse, and based on the severity, the stressors can affect gut healthby disrupting epithelial barrier function thus making the gut a “leakygut.” A leaky gut permits increased leakage of luminal commensalbacterial endotoxins (LPS, peptidoglycan) or pathogens into thesubmucosal location resulting in increased inflammatory cytokinesecretion (TNFα, IL-6, TGFβ, IL-8, etc) from intestinal epithelial cellsand immune cells. Nutritional deprivation also severely impairs gutfunction including reduced mucus secretion, and shortened villi heightand crypt depth. Thermal stress can damage the intestinal epithelium andelicit enterocyte membrane damage or death alters villus/cryptstructure, impairs tight junction integrity and increases endotoxinlevels in the blood. Epithelial cells exposed to stress have shownincreased Hsp60 expression. Altogether, stressors affect growthperformance and make animals susceptible to various infectious agentswith a huge financial loss to the farmers. Antibiotics are often used infeed to control infections and to enhance growth performance; however,concerns for the onset of antibiotics resistance require an alternativeapproach.

Potential benefits of “Next Generation Bioengineered Probiotics (NGBP)”expressing Listeria adhesion protein (LAP) from a nonpathogenic Listeria(L. innocua) that binds to a mammalian cell receptor, Hsp60 on animalhealth:

-   -   LAP-expressing NGBP can be used as a feed supplement to help        improve gut health by increased colonization of probiotics by        specifically interacting with mucosal Hsp60.    -   NGBP can improve gut barrier function by exerting        anti-inflammatory response    -   Generally, probiotics are most effective when normal intestinal        homeostasis is perturbed, particularly during periods of stress.        Here NGBP with increased interaction with stressed mucosal cells        would be very effective in maintaining intestinal homeostasis.    -   Because of anti-inflammatory and anti-microbial response of        NGBP, antibiotics use in feed can be reduced or eliminated.    -   NGBP mediated improved gut health can also enhance growth        performance and body weight.

The present invention may be better understood in light of the followingnon-limiting compound examples and method examples.

LAP from Listeria innocua (non-pathogen) restored adhesion andepithelial translocation ability of the lap-deficient L. monocytogenesstrain to enterocytes. LAP from pathogenic Listeria (i.e., L.monocytogenes) shares 99.3% amino acid sequence with the LAP from anonpathogenic Listeria (L. innocua) (Bailey et al., 2017; Jagadeesan etal., 2010) (FIG. 1A). It is proposed that extracellular secreted LAP inthe pathogen, re-associates on the surface of the bacterium to aid inbacterial adhesion and translocation across the epithelial barrier(Jagadeesan et al., 2010). However, in L. innocua, LAP does not aid inadhesion to the intestinal epithelial cells possibly due to a defect inre-association of the protein on the surface of this bacterium(Burkholder et al., 2009; Jagadeesan et al., 2010). Here, we show thatthe lap of L. innocua strain F4248, cloned and expressed in thelap-deficient L. monocytogenes mutant strain (KB208LAP^(Lin)) (FIG. 1B)restored its adhesion to (FIG. 1D), and translocation across (FIG. 1E)the Caco-2 cell monolayers at levels similar to that of a lap-mutantstrain expressing LAP of L. monocytogenes (KB208LAP^(Lm)), or the L.monocytogenes WT (F4244, serovar 4b). These data provide a strongindication that LAP from L. innocua has similar adhesion characteristicsas the L. monocytogenes WT strain.

Lactobacillus casei expressing LAP of L. innocua reduced L.monocytogenes infection in Caco-2 cell and a mouse model. The lap ORF(2.6 kb) from both L. innocua and L. monocytogenes was cloned separatelyinto a Lactobacillus expression vector, pLP401T (Koo et al., 2012;Maassen et al., 1999), and the proteins were expressed on the wild-typeprobiotic Lactobacillus casei ATCC344 (LbcWT) and designatedLbcLAP^(Lin) and LbcLAP^(Lm), respectively. LAP expression in bothbioengineered Lactobacillus probiotics (BLP) strains was confirmed byimmunoblotting (FIG. 2A), immunofluorescence staining and flow cytometry(FIGS. 3A, 3B) using anti-LAP mAb. LAP induces epithelial barrierdysfunction and promotes L. monocytogenes translocation across themucosal membrane (Drolia et al., 2018); therefore, the BLP strains(LbcLAP^(Lin) and LbcLAP^(Lm)) were tested in the in vitro transwellsetup for their ability to traverse epithelial-barrier. Interestingly,none of the BLP strains showed any significant translocation while theL. monocytogenes WT strain as a control showed a significantly very hightranslocation (FIG. 2B). These data indicate probiotics' natural abilityto maintain the epithelial barrier integrity possibly supersedesLAP-induced epithelial barrier dysfunction (Bron et al., 2017; Pagniniet al., 2010).

Next, we examined if these BLP strains could prevent L. monocytogenesinteraction with the epithelial cells. BLP strain pre-exposed to Caco-2cell line significantly lowered L. monocytogenes adhesion to (FIG. 2C)and translocation across the epithelial monolayer in transwell (FIG.2D), while the LbcWT pre-exposure did not show any significant reductionin L. monocytogenes translocation. A plasmid-vector control strainwithout the lap insert (LbcWT^(VC)) produced similar results as LbcWT,thus dismissing any extraneous anti-listerial effects that could becontributed by the virgin plasmid. In addition, none of the probioticstrains produced any anti-listerial compounds analyzed by the agarwell-diffusion assay (FIG. 3C) thus ruled out the involvement of anybacteriocin-like inhibitory substance. Next, we hypothesized that theBLP-mediated inhibition of L. monocytogenes interaction with theepithelial cells could be facilitated by direct binding of L.monocytogenes cells to BLP since BLP expresses LAP, and the LAP has anatural affinity towards its own surface (Burkholder et al., 2009;Jagadeesan et al., 2010). Therefore, we examined the interaction betweenBLP and L. monocytogenes cells, if any, in a suspension culture. Using aListeria-specific immunomagnetic bead (IMB; Invitrogen) capture system,we showed that L. monocytogenes WT bound strongly with the BLP cells,while a significantly reduced level with the LbcWT suggesting that BLPinteraction (aggregation) with L. monocytogenes was mediated by the LAP(FIG. 2E). Collectively, these data suggest that pre-occupation of theepithelial surface by LAP-expressing BLP can competitively exclude L.monocytogenes from interacting with the epithelial cells.

The prophylactic effect of BLP feeding on listeriosis in mice wasinvestigated using 8-10 weeks old female A/J mice that are highlysensitive to listeriosis (Czuprynski et al., 2003) in four experimentaltrials conducted over 5 years. Before the mice feeding experiment,probiotics survival in the simulated gastric fluid (SGF) and simulatedintestinal fluid I (SIF-I) and II (SIF-II) were ensured by platecounting (FIGS. 3D-3F). Live/dead staining using carboxyfluoresceindiacetate succinimidyl ester (cFDA) and propidium iodide (PI) alsoconfirmed probiotics survival in gastric fluids (FIG. 3G) and Westernblot showed LAP expression on BLP strains while grown in SIF-II (FIG.3H).

Freshly grown probiotics bacteria were supplied daily in 50 ml drinkingwater per mouse (probiotic viability was maintained at about 4×10⁹CFU/ml) for 10 days before an oral challenge with L. monocytogenes F4244(serovar 4b) strain (5×10⁸ CFU/mouse) (FIG. 4A). All probiotic-fedanimals maintained a constant body weight during the entire study, evenafter the challenge with L. monocytogenes strain on day 10. However, theanimals that did not receive any probiotics, but were challenged with L.monocytogenes lost >15% body weight (FIG. 4B). BLP-fed mice appearedhealthy and continued to feed and drink even after L. monocytogeneschallenge, while the control animals without any probiotics or animalsreceiving the LbcWT but were challenged with L. monocytogenes, appearedill (FIG. 5). The sick animals displayed ruffled hair, recumbency,reduced responsiveness to external stimuli, and reduced feed intake.

Animals were sacrificed at 24 and 48 h post-infection (pi) in Trial 1(n=60) and after 48 h pi in Trial 2 (n=30). L. monocytogenes counts inthe liver, spleen, MLN, kidneys, blood, intestine, and feces weredetermined (FIGS. 4E-4K). Irrespective of the tissues or organsexamined, the LbcWT feeding resulted in a meager 0-1 log CFU/mousereduction of L. monocytogenes counts. (FIG. 4E-4K). Astonishingly, theBLP-fed mice showed a reduction of L. monocytogenes counts by 1.5-3 log(up to 99.9%) after 24 h and 3.5-5 log (up to 99.999%) 48 h pi in liverand spleen of half the test population while L. monocytogenes wasundetectable in the remainder of mice. L. monocytogenes was alsoundetectable in blood and the kidney of BLP-fed mice (FIG. 4H, 4I). Nobackground Listeria was detected from any mice that received only theprobiotics or no probiotics at all.

Intestinal colonization and fecal shedding of L. monocytogenes inprobiotic-fed mice were also examined. BLP feeding also significantlyreduced L. monocytogenes colonization in the intestine (FIG. 4J) andfecal shedding (FIG. 4K), compared with that of the LbcWT-fed mice.Total lactic acid bacteria (LAB) counts in the intestine, and feces ofmice were relatively constant irrespective of the bacterial treatments(FIG. 4C). While the LbcWT and the BLP colonization in the gut(intestine and feces) were maintained at about 4.5 log and 5.5 logCFU/mouse, respectively, when intestinal samples were analyzed 48 h pi(FIG. 4D). Collectively, these data demonstrate that bioengineeredprobiotics were maintained in the intestine of mice for the duration ofthe study and protected mice from the extra-intestinal spread of L.monocytogenes.

Bioengineered probiotics protected gut barrier integrity. We havedemonstrated in previous studies that LAP induces epithelial barrierdysfunction and promotes of L. monocytogenes translocation in both invitro cell culture (Burkholder and Bhunia, 2010; Kim and Bhunia, 2013)and in vivo mouse model (Drolia et al., 2018). Countering this effect,probiotics are known to maintain epithelial tight junction integritythrough the immunomodulatory effect which is orchestrated by NF-kB andthe secretion of proinflammatory cytokines such as TNFα, IL-10, IL-6(Ahrne and Hagslatt, 2011; Pagnini et al., 2010; Zareie et al., 2006).First, we examined if the BLP were able to maintain the intestinalepithelial integrity thereby preventing L. monocytogenes translocationto extra-intestinal sites. Epithelial permeability was assessed inCaco-2 cell monolayers by monitoring the diffusion of FD4 from apical tobasolateral side (FIG. 6A), and by measuring transepithelial electricalresistance (TEER) in a trans-well set up (FIG. 6B). Caco-2 cellspre-treated with or without LbcWT for 24 h followed by L. monocytogeneschallenge for 2 h resulted in a very high FD4 permeability (61-64%change) compared to the control, while LbcLAP^(Lm) or LbcLAP^(Lin),pre-treatment for 24 h substantially reduced FD4 translocation followingL. monocytogenes infection resulting in only 22% change compared to theuntreated control (FIG. 6A). Likewise, BLP (LbcLAP^(Lm) or LbcLAP^(Lin))pre-exposure followed by L. monocytogenes caused only 2.3-6.9% change inCaco-2 TEER values while L. monocytogenes alone caused a 17% change(FIG. 6B).]

Gut permeability was also assessed in BLP-fed mice by monitoring thelevels of FD4 in serum and urine (Drolia et al., 2018). BLP-fed micechallenged with L. monocytogenes were orally administered with the FD44-5 h prior to sacrifice (Drolia et al., 2018). Animals (FIGS. 2A-2F)that did not receive any probiotics, but were challenged with L.monocytogenes had an FD4 level at 3.5±0.3 μg/ml in sera (FIG. 6C) and83.3±13.5 μg/ml in urine (FIG. 6D). In LbcWT-fed animals, the FD4 levelsin sera and urine were 1.82 μg/ml and 18.0 μg/ml, respectively, whilethe FD4 levels were 2.6 μg/ml and 46.9 μg/ml, after L. monocytogeneschallenge. In contrast, the FD4 levels in both sera and urine in animalsthat received BLP with or without L. monocytogenes challenge hadsubstantially lower FD4 (about 1.9 μg/ml in sera and 13.2-22.4 μg/ml inurine) equivalent to that of the control mice that did not receiveeither bacterium. These data clearly demonstrate that probioticsespecially the LAP-expressing BP were able to attenuate L.monocytogenes-mediated epithelial dysfunction in a mouse model.Transmission electron microscopy (TEM) also showed epithelial tightjunction opening in ileal tissue sections of mice fed with Lm but notwith bioengineered probiotic followed by Lm as examined by transmissionelectron microscopy (TEM).

LAP-mediated epithelial barrier dysfunction is governed bymislocalization of epithelial junction proteins, claudin-1, occludin,and E-cadherin (Drolia et al., 2018). In Caco-2 cells, L. monocytogenesWT alone or Caco-2 pre-treated with probiotics significantly decreasedmembrane localization of claudin-1, occludin, and E-cadherin analyzed byWestern blotting (FIG. 7A) and the corresponding transcripts inagreement with our previous study (Drolia et al., 2018). Pre-treatmentwith the BLP prevented L. monocytogenes-mediated claudin-1, occludin andE-cadherin mislocalization. Confocal immunofluorescence microscopyconfirmed destabilization of the cell junction architecture as ZO-1disruption by L. monocytogenes was pronounced with a discontinuous cellmembrane boundary, which was not seen when the Caco-2 cells werepre-treated with the BLP (FIG. 7B). E-cadherin and claudin weresequestered in L. monocytogenes-treated Caco-2 cell cytoplasm but wasnot seen in the BLP pre-treated cells (FIG. 7B). Similar results wereseen in mice ilea where BLP-fed mice maintained intact claudin-1,occludin and E-cadherin even after challenging with L. monocytogenes(FIG. 7C). Taken together, these data indicate that both BLP strains,but not the wild-type probiotics maintained the tight junction integrityand thus prevented L. monocytogenes movement across the epithelial cellbarrier.

Ileal tissue histology and innate immune response to bioengineeredprobiotic. Ileal tissue sections from mice collected at 48 h pi werefirst examined for inflammation after hematoxylin and eosin staining.Overall, the inflammation due to L. monocytogenes infection in 48 h piwas subtle (FIGS. 8A-8B). Ileal tissues of untreated control mice hadcylindrical villi with relatively few lymphocytes in the lamina propria.Goblet cells (10% of the villous epithelium) and Paneth cells weremostly confined to the intestinal crypts. The remaining cells of thevillous epithelium were enterocytes. The mice that did not receive anyprobiotics, but were challenged with L. monocytogenes had mildlyincreased numbers of goblet cells at 24 h than the mice received onlythe LbcWT (FIGS. 8C-8D) in the villous epithelium with neutrophilinfiltration at the base of the villous lamina propria. The BLP-fed micechallenged with L. monocytogenes showed the highest averagehistomorphological score, and higher goblet cell counts; however, theenterocytes remained intact with no sign of apparent necrosis (FIGS.8A-8D).

Mammalian Hsp60 activates innate immune response (Chen et al., 1999;Pockley, 2003). Earlier, we observed that L. monocytogenes infectioninduced membrane Hsp60 expression, which subsequently facilitatedenhanced LAP-mediated L. monocytogenes translocation (Burkholder andBhunia, 2010; Drolia et al., 2018) by breaching an innate immune systemin the mouse. Therefore, we examined the Hsp60 expression in the mouseileal sections. Hsp60 expression was pronounced and uniformlydistributed on the villous epithelial cells of mice that did not receiveany probiotics but challenged with L. monocytogenes for 48 h (FIG. 8E).Expression of Hsp60 was lower in all probiotic-fed mice but enhancedwhen challenged with L. monocytogenes. A similar trend was observed whenthe levels of hsppdl (hsp60) transcripts were analyzed in the ilealtissue samples (FIG. 8F). These data demonstrate that BLP was able todampen the Hsp60 expression, however, after the L. monocytogeneschallenge, Hsp60 expression increased in epithelial cells, which mayhelp the host cells to defend against the infection through a mechanismwhich requires further investigation.

Immunomodulatory and anti-inflammatory effects of probiotic feeding inmice. Probiotic bacteria modulate the immune response and maintainimmune homeostasis via activation of NF-κB and production of epithelialTNF-α (Cross, 2002; Pagnini et al., 2010). Moreover, both TNF-α and IL-6increase epithelial barrier permeability through activation of NF-κB (Maet al., 2004). Earlier, we have shown that LAP of L. monocytogenesstimulates NF-κB, produces epithelial TNF-α and IL-6 and increasesepithelial permeability by dysregulating epithelial junctional proteins(Drolia et al., 2018). Here, we observed that the BLP strains loweredapproximately 2-fold NF-κB activity in a Luciferase reporter RAW (murinemacrophage) cell line compared to that of L. monocytogenes WT orLPS-treated control cells (FIG. 9A). Confocal immunostaining of ilealtissue samples also showed increased translocation of P-p65 and p65 intothe nucleus by L. monocytogenes WT but reduced levels in BLP-pretreatedmice indicating that BLP stimulates NF-kB (FIG. 9B). Furthermore, TNF-αand IL-6 levels were increased substantially in the murine ileal tissueextracts (FIGS. 9C-9D) from L. monocytogenes infected mice withoutprobiotic feeding, while the levels were equivalent to that of theuninfected controls when fed with the BLP strains.

To assess the state of systemic immune response in BLP-fed mice, levelsof several cytokines in the pooled sera from the three animals withineach treatment group were analyzed using a semi-quantitative immunoblotarray. Strong IL-6 and MCP-1 response were observed in animals that wereinfected with L. monocytogenes without any pre-exposure to probiotics;however, both the wild-type probiotic and BLP exposure significantlydampened these cytokines in L. monocytogenes-infected mice. In contrast,levels of G-CSF was very high in sera after L. monocytogenes challenge,irrespective of the probiotics used. Serum TNF-α level was undetectableirrespective of the treatments, possibly the array could not detecttrace amounts.

Probiotic bacteria also influenced cellular immune response to L.monocytogenes infection as seen in the spleen by flow cytometry andcytology. Among the splenic CD4⁺ (FIG. 9E) and CD8α⁺ (FIG. 9F) T cellpopulations, only CD8α⁺ counts showed a strong response in micechallenged with L. monocytogenes in the absence of any probiotic, whilethe counts were lower in BLP-primed mice following L. monocytogeneschallenge. L. monocytogenes infection also increased splenic CD11c⁺(dendritic cell) counts irrespective of the type of probiotics used(FIG. 9G).

Cytological imprints from splenic cross-sections did not reveal anyobvious lymphoid hyperplasia in control animals while L. monocytogenesinfection resulted in significant neutrophil and macrophage infiltration(FIG. 9I). No cytological evidence of inflammation was apparent in anyprobiotic-fed animals. Interestingly, BLP-fed animals followed by L.monocytogenes challenge showed moderate-to-marked inflammation withincreased infiltration of macrophages and neutrophils (FIG. 9I). Blindedcytology scoring also confirmed such observation (FIG. 9H). Theimmunomodulatory and anti-inflammatory effects of probiotics and reducedListeria counts in spleen (FIG. 4F) strongly suggest that BLP positivelyinfluenced cellular immune response for efficient clearance of L.monocytogenes from extra-intestinal sites.

Immunomodulatory effect of probiotic was also assessed in ileal tissuesby immunostaining of ileal tissue sections with T-cell markers,anti-CD3⁺; anti-CD8⁺ and CD4⁺ FoxP3⁺ antibodies which revealedsignificant differences in total T-cell counts between the control andbioengineered probiotic-fed mice as shown in FIGS. 10A-10D.Bioengineered probiotics (BLP) enhanced the regulatory T cell (CD4⁺FoxP3⁺) response while cytotoxic (CD8⁺) cell counts were low. Thissuggests that bioengineered probiotic was able to prime the immunesystem, which possibly helped eliminate invaded L. monocytogenes cellsand maintain tight junction integrity to prevent bacterial passage orincreased clearance of pathogens that were able to cross the epithelialbarrier, especially from the BLP-fed mice where most animals showedreduced systemic infection (FIGS. 4A-4K).

FIG. 11 shows survival of mice (A/J strain) supplied with or withoutprobiotics for ten days in drinking water followed by oral infectionwith L. monocytogens (n=at least 10 per group). Mice fed withLactobacillus casei (probiotics) expressing the LAP protein (LbcLAPLin)showed significantly (**P<0.01, Kaplan-Meier log-rank test) highersurvival compared to that of LbcWT.

FIG. 12. Comparison of the amino acid sequence of LAP (Listeria adhesionprotein) from different strains of Lm and Lin obtained from the NCBIdatabase.

FIG. 13. Confirmation of Bioengineered Lactobacillus probiotic (BLP)strains expressing LAP from Listeria innocua (LbcLAP^(Lin)) or L.monocytogenes (LbcLAP^(Lm)) by Western blotting (left panel) andconfocal imaging (right panel; arrows)

FIG. 14. BLP strains expressing LAP from Listeria innocua (LbcLAP^(Lin))or L. monocytogenes (LbcLAP^(Lm)) prevent Lm interaction in vitro in aCaco-2 cell culture model. Increased inhibition of Lm adhesion (left,n=6), invasion (middle, n=6) and translocation (right, n=6) by the BLPstrains after 24 h exposure to Caco-2 cells.

FIG. 15. Schematics showing mouse experiment protocol.

FIG. 16. BLP strains expressing LAP from Listeria innocua (LbcLAP^(Lin))or Lm (LbcLAP^(Lm)) prevent Lm induced weight loss in mice. Normalizedmouse body weight (mean±SD, n=5) on day 0, 5, 10, and 12.

FIG. 17. BLP strains expressing LAP from Listeria innocua (LbcLAP^(Lin))or Lm (LbcLAP^(Lm)) show increased intestinal colonization. IncreasedBLP counts in the intestinal content of mice (n=5) on days 10, 11, and12.

FIG. 18. BLP strains expressing LAP from Listeria innocua (LbcLAP^(Lin))or Lm (LbcLAP^(Lm)) prevent Lm intestinal infection in a mouse model.Reduced Lm burdens in the intracellular location in the ileum (left,n=6), cecum (middle, n=6), and colon (right, n=6).

FIG. 19. BLP strains expressing LAP from Listeria innocua (LbcLAP^(Lin))or Lm (LbcLAP^(Lm)) prevent Lm systemic infection in a mouse model.Reduced Lm burdens in the liver (left), spleen (right) (left and right,n=5 at 24 h post infection (hpi); and n=17, 14, 13, 14 at 48 hpi), MLN(center, n=11, 9, 8, 9, for each group, respectively),

FIG. 20. BLP strains expressing LAP from Listeria innocua (LbcLAP^(Lin))or Lm (LbcLAP^(Lm)) prevent Lm systemic infection in a mouse model.Reduced Lm burdens in the blood (right, n=5 at 24 hpi; and n=11, 8, 8,8, at 48 hpi for each group, respectively) and kidney (left, n=6, 3, 3,3, for each group, respectively).

FIG. 21. BLP strains expressing LAP from Listeria innocua (LbcLAP^(Lin))or Lm (LbcLAP^(Lm)) prevent Lm intestinal penetration in a mouse model.Micrographs of ileal (left) villi immunostained for ZO-1 (brown) and Lm(red, arrows) and counterstained for nucleus (blue) at 48 hpi. Bars, 10μm. The boxed areas were enlarged. Bars, 1 μm. Lm counts (mean±SEM) inileal lamina propria (LP; right panels). Dots represent an average of 25villi from a single mouse, four mice/group, n=100 villi. Lm is observedin the LP (arrows) in naive or LbcWT-treated mice but confined in thelumen (arrows) in BLP-treated mice (Lbc LAP^(Lin) and Lbc LAP^(Lm)).

FIG. 22. BLP (LbcLAP^(Lin)) prevents lethal L. monocytogenes infectionin mice. Increased survival of BLP-treated mice (LbcLAP^(Lin)) at LD₅₀dose. *p<0.05 Kaplan-Meier log-rank test.

FIGS. 23A-23B. BLP colonization and persistence in the intestine limitsLm translocation despite discontinuous administration. Schematics (FIG.23A) showing mouse experiment protocol: Mice were treated with L. casei(LbcWT) or BLP (LbcLAP^(Lm)) strain supplied in drinking waterreplenished daily (4-8×10⁹ CFU/ml) for 10 days (0-9 days) and thenchallenged with Lm F4244 (˜5×10⁸ CFU/animal) on day 10, 15 and 20. (FIG.23B) BLP-mediated reduced Lm burdens at 48 hpi in the intracellularlocation in the ileum (left), cecum (center), and colon (right) (allbottom panels, gentamicin resistant CFU), in mice on day 12

FIG. 24. BLP displays increased co-aggregation with Lm. Increasedco-aggregation of BLP (left panel, LbcLAP^(Lin,) and LbcLAP^(Lm))strains in co-incubated suspensions containing equal numbers of BLP+Lmcells (n=4) captured via Listeria-specific immunomagnetic beads (IMB)but not with BLP+L. innocua (Lin, n=6) cells or L. casei (alone) (n=10).Micrographs (right panel) showing co-aggregated BLP cells (arrows) withIMB-captured Lm cells expressing GFP (b). Bars, 1 μm.

FIG. 25. BLP (LbcLAP^(Lin) and LbcLAP^(Lm)) forms increased biofilm onplastics. Increased biofilm formation (Abs 595 nm, mean±SEM) of BLPstrains as measured by crystal violet staining in monoculture andco-culture with grown in microtiter plates. Images (top panel) showcrystal violet stained biofilms of representative wells for eachtreatment. Data represent three independent experiments obtained fromn=6 independent microtiter plate wells.

FIG. 26. BLP (LbcLAP^(Lin) and LbcLAP^(Lm)) forms increased biofilm onmouse colonic villi (right two panels)

FIG. 27. BLP prevents Lm from causing intestinal barrier damage in amouse model. Representative H&E-stained micrographs (bars, 25 μm) (left)and the histological score (right, each point represents an individualmouse) of ileal tissue sections from control (mock-treated) uninfectednaïve mice or L. casei-treated (10 days, LbcWT or BLP) pre orpost-Lm-challenge at 48 hpi (n=9, 7, 7, 9, 9, 9, 10, 11 mice for eachgroup, respectively). Arrows point to the loss of villous epithelialcells and increased polymorphonuclear and mononuclear cells infiltratingthe base of the villous lamina propria in naïve (naive+Lm) and LbcWT-treated mice (LbcWT+Lm) at 48 hpi.

FIG. 28. BLP prevents Lm from causing intestinal barrier loss bymaintaining mucus-producing goblet cells in mice. Representativeimmunohistochemical micrographs of the ileum stained for Muc2 (left,brown), nuclei (blue) from control (mock-treated) uninfected naïve miceor L. casei-treated (10 days, LbcWT or BLP) pre or post-Lm challenge at48 hpi. Bars, 10 μm. Quantification of Muc2 (right)-positive cells, eachpoint represents an individual mouse, 4 mice per group, n=100 villi.Arrows point to increased numbers of Muc2 (left) in BLP treated mice(pre- or post-Lm challenge).

FIG. 29: BLP blocks Lm from causing disturbance of intestinal epithelialcell-cell junctional integrity. FITC conjugated 4 kDa dextran (FD4) gutpermeability of control (mock-treated) naïve uninfected mice or L.casei-treated (10 days, LbcWT or BLP) pre- or post-Lm challenge (48 hpi)in serum (c) and urine (d). Each point represents an individual mouse.Data represent mean±SEM of n=3 mice for all groups except Lm group, n=5mice.

FIG. 30. BLP blocks Lm from causing disturbance of intestinal epithelialcell-cell junctional integrity. Immunofluorescence micrographs of theileal tissues showing increased expression of MLCK and P-MLC (green;arrows) and mislocalization (intracellular puncta) of claudin-1 (green;arrows), occludin, and E-cadherin (red; arrows) in naïve orLbcWT-treated (10 days) but baseline expression of MLCK and P-MLC andintact localization of occludin, claudin-1 and E-cadherin in BLP-treatedmice (10 days) at 48 hpi, relative to uninfected naïve mice. Nuclei;DAPI, blue. Images are representative of five different fields from n=3mice per treatment. Bars, 10 μm. LP, Lamina Propria.

FIG. 31: BLP prevents Lm-induced NF-κB (p65) activation and modulatescytokine production and immune cells to maintain intestinal immunehomeostasis. Immunofluorescence micrographs of the ileal tissues showingdecreased nuclear localization of p65 (green) in BLP-treated mice (10days) at 48 hpi. Nuclei; DAPI, blue. Arrows indicate the nuclearlocalization of p65 in IEC of naïve or LbcWT-treated (10 days) mice at48 hpi. The right panels show the quantified results (mean±SEM) of p65nuclear positive IEC. Each point represents an average of 15 villi froma single mouse, 4 mice per group, n=60 villi. Bars, 10 μm.

FIG. 32: BLP modulates the cytokine TNFα to maintain intestinal immunehomeostasis. ELISA showing decreased TNFα (n=6 mice for all groupsexcept LbcWT and LbcLAP^(Lin)+Lm group; n=5 and 4 mice, respectively),in the ileal tissues of BLP-treated (10 days) mice, relative to naïve orLbcWT-treated (10 days) at 48 hpi.

FIG. 33: BLP modulates the cytokine IL-6 to maintain intestinal immunehomeostasis. ELISA showing decreased IL-6 (n=6 mice for all groupsexcept LbcWT and LbcLAP^(Lin)+Lm group; n=5 and 4 mice, respectively),in the ileal tissues of BLP-treated (10 days) mice, relative to naïve orLbcWT-treated (10 days) at 48 hpi

FIG. 34: BLP modulates the cytokine IFNγ to maintain intestinal immunehomeostasis. ELISA showing increased IFNγ (n=4 mice for all groups), inthe ileal tissues of BLP-treated (10 days) mice, relative to naïve orLbcWT-treated (10 days) at 48 hpi

FIG. 35: BLP modulates the cytokine IL-10 to maintain intestinal immunehomeostasis. Graph showing increased IL-10⁺ cells quantified (mean±SEM)from immunostained ileal tissues (of BLP-treated (10 days) mice pre- orpost-Lm challenge at 48 hpi. Each point represents an average of 25villi from a single mouse, 4 mice per group, n=100 villi.

FIG. 36: BLP modulates the cytokine TGFβ⁺ to maintain intestinal immunehomeostasis. Graph showing increased TGFβ⁺ cells quantified (mean±SEM)from immunostained ileal tissues of BLP-treated (10 days) mice pre- orpost-Lm challenge at 48 hpi. Each point represents an average of 25villi from a single mouse, 4 mice per group, n=100 villi.

FIG. 37: BLP modulates CD4⁺ T-cell populations for immunomodulation.Representative immunohistochemical micrographs of ileal tissues showingincreased CD4⁺ cells (left, brown, arrows), in BLP-treated mice (10days) mice pre or post-Lm challenge at 48 hpi, relative to naïve orLbcWT-treated (10 days). Bars, 10 μm. Quantification of CD4⁺ cells(right), Each point represents an average (mean±SEM) of 25 villi from asingle mouse, 4 mice per group, n=100 villi.

FIG. 38: BLP modulates FOXP3⁺ T-regulatory cells populations forimmunomodulation. Representative immunohistochemical micrographs ofileal tissues showing increased FOXP3⁺ T-regulatory cells (left, brown,arrows), in BLP-treated mice (10 days) mice pre or post-Lm challenge at48 hpi, relative to naïve or LbcWT-treated (10 days). Bars, 10 μm.Quantification of FOXP3⁺ T-regulatory cells (right), Each pointrepresents an average (mean±SEM) of 25 villi from a single mouse, 4 miceper group, n=100 villi.

FIG. 39: BLP modulates CD11c⁺ dendritic cells populations forimmunomodulation. Representative immunohistochemical micrographs ofileal tissues showing increased CD11c⁺ dendritic cells (left, brown,arrows), in BLP-treated mice (10 days) mice pre or post-Lm challenge at48 hpi, relative to naïve or LbcWT-treated (10 days). Bars, 10 μm.Quantification of CD11c⁺ cells (right), Each point represents an average(mean±SEM) of 25 villi from a single mouse, 4 mice per group, n=100villi.

FIG. 40: BLP modulates natural-killer NKp46⁺ immune cell populations forimmunomodulation. Representative immunohistochemical micrographs ofileal tissues showing increased NKp46⁺ cells (left, brown, arrows) inBLP-treated mice (10 days) mice pre or post-Lm challenge at 48 hpi,relative to naïve or LbcWT-treated (10 days). Bars, 10 μm.Quantification of NKp46⁺ cells (right), Each point represents an average(mean±SEM) of 25 villi from a single mouse, 4 mice per group, n=100villi.

FIG. 41. Schematics showing the mechanism of BLP-mediated protectionagainst listeriosis. The BLP prevents Lm Infection by three mechanisms(i) Competitive exclusion, (ii) improved intestinal barrier function,and (iii) contact-dependent immunomodulation.

We also measured the levels of secretory IgA (sIgA) in the ileal mucussamples, and the total sIgA levels for probiotic-fed mice wereconsiderably higher than the control animals; however, there were nodifferences in LbcWT and the BLP-fed mice indicating probiotics naturalability to induce sIgA production (Bakker-Zierikzee et al., 2006). Wealso could not detect any LAP-specific antibody in the pooled mice serafrom either LbcWT or BLP-fed mice. This provides evidence againstsubmucosal translocation of LAP-expressing probiotics, which is inagreement with Caco-2 transwell data (FIG. 2B). However, the sera fromprobiotic-fed mice that were challenged with L. monocytogenes for 48 hexhibited a very faint antibody reaction with LAP, for which there is nospecific explanation. Nevertheless, these data demonstrate thatprobiotics-mediated humoral immune response, especially the sIgA mayalso affect L. monocytogenes interaction with intestinal epithelialcells during the early phase of infection (Mantis et al., 2011).

Bioengineered probiotic feeding also increased survival of mice after L.monocytogenes challenge. Mice were fed with probiotic bacteria for 10days and then challenged with lethal dosage of L. monocytogenes (2×10⁹CFU/mouse). Mice survival was examined over 10 days. Over 82% mice frombioengineered probiotic (LbcLAP^(Lin))-fed mice group survived while 60%and 50% mice survived that received LbcWT and no probiotic control(naïve), respectively (FIG. 11). This study clearly indicates thatLAP-expressing bioengineered probiotic bacteria can prevent fatalinfection caused by L. monocytogenes.

Listeria monocytogenes is an invasive opportunistic intracellular humanpathogen. It is ubiquitous and is transmitted primarily through foodresulting in numerous fatal and costly outbreaks that are associatedwith consumption of contaminated cheese, ice cream, fish, ready-to-eatmeats, and produce (cantaloupe, apples, sprouts, spinach). Besidespregnancy, immune suppressed conditions in the elderly, and malignancy,organ transplant and HIV-AIDs patients are also highly vulnerable(Schuchat et al., 1991). The case fatality rate of listeriosis is 19%.Currently, there is no preventive vaccine against listeriosis except forgeneral precautionary guidelines outlined by the CDC that includethorough cooking of meat, safe food handling practices and avoidance ofthe FDA designated high-risk foods, such as frankfurters, soft cheesesmade with unpasteurized milk, pate, and smoked fish. Therefore,prophylactic intervention strategies for the high-risk population fromlisteriosis would have a greater public health impact. One of thepromising alternatives to the use of antibiotics in prophylaxis ortherapy is the utilization of probiotic microbes (Amalaradjou andBhunia, 2012; Sanders et al., 2014). Probiotic microbes also producemetabolites and macromolecules promoting gut health by modifyingcytokine production and enhancing gut barrier function (Bron et al.,2017; Cho et al., 2014; Salminen et al., 2010). Probiotic microbes canprevent/alleviate chronic inflammatory bowel disease, colorectal cancer,metabolic disorders and obesity, and osteoporosis (Amalaradjou andBhunia, 2012; Azcarate-Peril et al., 2011; Ly et al., 2011). Probioticsare also used in pre-term neonates to allow early colonization withbeneficial microbes (Deshpande et al., 2011), and increased sIgAsecretion in the gut (Bakker-Zierikzee et al., 2006). Among thedifferent probiotic bacteria used, Lactobacillus species is most commonbecause of their ability to survive, colonize and modulate the immunesystem in the gut, and are generally safe (Amalaradjou and Bhunia,2012). Earlier, Corr et al. (Corr et al., 2007) showed that bacteriocinproducing Lactobacilli could control listeriosis in a mouse model.However, probiotics approach has been ineffective or has had limitedsuccess against listeriosis (Culligan et al., 2009; Koo et al., 2012).To overcome such limitations, we bioengineered a probiotic Lactobacilluscasei strain to prevent Listeria interaction with the epithelial cellsin the intestinal tract and subsequent extra-intestinal dissemination.

We have shown previously that LAP plays an important role duringearly-phase of infection (within 24-48 h), promoting translocation of L.monocytogenes across the epithelium in mice (Burkholder et al., 2009;Drolia et al., 2018). The LAP lacks a leader sequence thus the bacterialsecretory system, SecA2 helps LAP to secrete to the extracellular milieuand for surface display (Burkholder et al., 2009; Mishra et al., 2011).The LAP from L. monocytogenes bears high sequence similarity to the LAPfrom L. innocua (non-pathogen) and the L. innocua LAP is unable tore-associate on its own surface possibly due to the lack of a surfaceanchoring molecule (Jagadeesan et al., 2011; Jagadeesan et al., 2010).This defect probably prevents L. innocua from translocating through theepithelial paracellular route (Burkholder and Bhunia, 2010).Interestingly, the L. innocua LAP fully restored epithelialtranslocation ability in a lap-deficient L. monocytogenes strain in acell culture model (this study). This raised an intriguing question; canthe LAP from L. innocua expressed on probiotic Lactobacillus preventlisteriosis in a mouse model? A/J mice are highly sensitive tolisteriosis due to C5 complement deficiency (Czuprynski et al., 2003;Jagannath et al., 2000); therefore, these animals should be ideal forstudying the prophylactic effect of BLP against listeriosis.

Incredibly, both bioengineered Lactobacillus casei expressing LAP^(Lm)or LAP^(Lin) were able to prevent L. monocytogenes disseminationsubstantially (up to 5 log or 99.999% reduction) to extra-intestinaltissues and organs and the mice appeared healthy when sacrificed at 48 hpi. Both LbcWT and BLP were maintained in the gut during the 10 daysfeeding trials and they were not detected in any extra-intestinaltissues upon sacrifice implying that either they did not cross theintestinal barrier or the translocated BLP were cleared immediately bythe local immune system. Blood sera also did not reveal any noticeableLAP-specific antibody response suggesting that the LAP antigen may nothave disseminated systemically.

Two plausible mechanisms for BLP-mediated protection are postulated: (i)Prevention of L. monocytogenes interaction with the intestinalepithelial cells by BLP via preoccupation of the intestinal niche, andsubsequent binding to L. monocytogenes, and (ii) activation of theimmune system for increased clearance of the translocated pathogens. Ourresults also indicate that the BLP prevented L. monocytogenesdissemination by maintaining epithelial tight junction integrity as thepreservation of the cytoskeleton and tight junction barrier integrity iscritical for modulating paracellular and transcellular bacterialdiffusion (Pagnini et al., 2010; Zhou et al., 2010). Mislocalization ofepithelial junctional proteins, occludin, claudin-1 and E-cadherin inthe ileal tissues of the mice was evident in L. monocytogenes infectedmice and the LbcWT-fed groups, while the cell junction architectureremained intact in animals fed with BLP followed by L. monocytogenesinfection.

Probiotic bacteria exert immunomodulatory effect (Ng et al., 2009) andpromote gut health through stimulation of epithelial innate immunity bystimulating local production of TNF and activation of NF-kB (Pagnini etal., 2010). In agreement with a previous report (Rothe et al., 1993),here we also observed L. monocytogenes mediated high levels of TNF-α andIL-6 in the ileal tissue homogenates and IL-6 level in the sera. Indeed,activation of NF-kB results in elevated levels of TNF-α and IL-6, whichfacilitate gut epithelial barrier destabilization (Drolia et al., 2018;Ma et al., 2004). In our previous report, LAP induced epithelial IL-6and TNF-α production during L. monocytogenes infection throughactivation of NF-κB (Drolia et al., 2018), in this study, LAP-expressingBLP was able to dampen L. monocytogenes-mediated proinflammatorycytokine production despite moderate activation of NF-kB. This suggests,perhaps BLP helped maintain epithelial immune homeostasis thus was ableto counteract L. monocytogenes mediated inflammatory response. Duringinnate immunity, epithelial cells and monocytes secrete IL-6 whenstimulated by pathogen-associated molecular patterns (PAMPS) on specificpathogens that are recognized by pattern recognition receptors (PRR)including Toll-like receptors (TLRs) similar to LAP-Hsp60 interaction(Drolia et al., 2018). Previous in vitro studies using RAW264.7macrophages exposed to cell wall extracts of Bifidobacteriumadolescentis, B. longum, and Lactobacillus salivarius Ren enhancedphagocytic activity via increased production of IL-6 and TNF-α (Zhu etal., 2011). Oral gavage of mice with L. acidophilus and B. bifidumshowed increased reactive oxygen intermediates production and enhancedphagocytic activity in macrophages (Deepti and Vinod, 2014). A long-termconsumption of probiotic has shown to enhance innate immunity andproduction of IL-1, IL-1β, IL-6, IL-10, IL-12, IL-18, INF-γ, and TNF-αby monocytes and DC (Cross, 2002; Niers et al., 2005).

Cell-mediated immunity especially the CD8⁺ T-cell response is criticalfor controlling systemic L. monocytogenes infection (Huleatt et al.,2001). Here we also observed increased cell counts with CD8α⁺ marker inspleen in the L. monocytogenes infected control group 48 h pi, while theopposite trend in BLP-fed animals. CD8α⁺ cells represent both cytotoxicT-cells and a subset of dendritic cells, and both are requisite forefficient splenic infiltration during intravenous administration of L.monocytogenes in mice (Edelson et al., 2011). The concomitant markedincrease in neutrophils, macrophages and dendritic cells (CD11c⁺ andspleen cytology data) in the spleen with L. monocytogenes infection inthe BP-fed groups above the control infection group suggests thatperhaps the BP strains serve to prime the innate immune system. As such,increased phagocyte infiltration may lead ultimately to improvedpathogen clearance without the need for CD8⁺ T-cells. Prophylactic oraladministration of L. casei CRL431 has positively influenced neutrophilresponse to a nasally inoculated Streptococcus pneumoniae, demonstratinga potentially important link in mucosal immunity between different organsystems (Villena et al., 2005). The total splenic CD4⁺ T cell populationdid not change in our study, but other T_(H) subtypes that maycontribute to the overall differential immune response were notmeasured. Probiotic microbe-induced sIgA response in ileal mucus in micepre-exposed to both LbcWT and the BLP, akin to previous studies(Bakker-Zierikzee et al., 2006; Sakai et al., 2014) suggesting that theprobiotic bacteria stimulate mucosal immune response (Mantis et al.,2011) against L. monocytogenes.

In summary, the wild-type probiotic strains tested are generallyineffective against L. monocytogenes infection (Koo et al., 2012);therefore, the bioengineered probiotic strains were made to preventlisteriosis in a mouse model. Our study has demonstrated that theLAP-expressing bioengineered Lactobacillus casei, including the LAP froma nonpathogenic Listeria, protected mice from L. monocytogenes infectionthrough colonization resistance, maintenance of gut permeability andtight junction stability, and immunomodulation. Such bioengineeredstrain can potentially prevent listeriosis in high-risk populations andat the same time promote health benefits inherent to probioticlactobacilli.

Materials and Methods

Bacterial strains, plasmids, and growth conditions. All Listeria specieswere grown in tryptic soy broth containing 0.5% yeast extract (TSBYE;Becton Dickinson, Sparks, Md.) or Luria-Bertani broth (LB, 0.5% NaCl, 1%tryptone peptone, and 0.5% yeast extract) at 37° C. for 16 to 18 h.Probiotic bacteria were cultured in deMan Rogosa Sharpe broth (MRS,Becton Dickinson) at 37° C. for 18-20 h. Lactobacillus casei ATCC 344wild-type (LbcWT) (a gift from Mike Miller, University of Illinois,Urbana) was used as a host to express LAP from L. innocua and L.monocytogenes. To recover this strain from fecal and intestinal samplesduring the animal study, a vancomycin-resistant strain of L. casei wasselected by sequentially culturing the bacterium in increasingconcentrations of vancomycin (300 μg/ml). Recombinant L. paracasei wasgrown under anaerobic conditions at 37° C. with erythromycin (2 μg/mL).The lap-deficient mutant L. monocytogenes strain KB208 was grown inTSBYE with erythromycin (10 μg/mL) at 42° C. KB208 expressing L. innocuaLAP was grown in TSBYE with erythromycin (5 μg/mL) and chloramphenicol(7 μg/mL) at 42° C.

Generation of bioengineered lactobacilli expressing LAP from L. innocuaand L. monocytogenes. The entire lap gene (2.6 kb) from L. innocua wasamplified by PCR and inserted into pLP401T (Pouwels et al., 2001) andelectrotransformed into L. casei ATCC 334 designated LbcLAP^(Lin) (L.casei AKB907) as described before (Koo et al., 2012). Likewise, lap genefrom L. monocytogenes was expressed in L. casei designated LbcLAP^(Lin)(AKB906). The bioengineered strains were maintained in MRS brothcontaining erythromycin (2 μg/ml) under anaerobic conditions at 37° C.The L. innocua lap gene was cloned into pMGS101, electrotransformed intoKB208, and designated LmKB208LAP^(Lin). To induce LAP expression, thebioengineered L. casei strains, were grown in modified MRS (1% w/vprotease peptone, 0.5% w/v yeast extract, 0.2% w/v meat extract, 0.1%v/v TWEEN® 80, 37 mM C₂H₃NaO₂, 0.8 mM MgSO₄, 0.24 mM MnSO₄, 8.8 mMC₆H₁₄N₂O₇ in 0.1 M potassium phosphate buffer, pH 7.0) supplemented withmannitol (1% w/v). LAP expression was verified by Western blotting,ELISA and immunofluorescence staining using anti-LAP mAb (Koo et al.,2012).

Growth characteristics of recombinant probiotics in artificialgastrointestinal fluids. The survival of probiotics exposed sequentiallyto the simulated gastrointestinal fluid (SGF) and simulated intestinalfluid (SIF-I and SIF-II), to simulate gastric phase, enteric phase 1 andenteric phase 2, respectively), over 6 h (2 h for each step) period wasmonitored (Buriti et al., 2010). SGF contained pepsin (3 g/L) and lipase(0.9 mg/L) (Sigma-Aldrich), pH 1.2-1.5 (adjusted using 1N HCl). BothSIF-I and SIF-II contained bile (bovine bile; 10 g/L, Sigma-Aldrich) andporcine pancreatin (1 g/L; Sigma-Aldrich), but SIF-I pH was 4.3-5.2 andSIF-II pH 6.7-7.5 (adjusted using alkaline solution; 150 ml of 1 N NaOH,14 g of PO₄H₂Na.2H₂O and deionized water up to 1 L). Overnight culturesof wild-type or BP were washed and resuspended in SGF (100 ml) andincubated at 37° C., with agitation (150 rpm for 2 h) (gastric phase),and bacterial counts were monitored every 30 min for 2 h. The cells fromSGF were pelleted down and transferred sequentially into SIF-I, andSIF-II, incubated each at 37° C. for 2 h to simulate the initial andfinal phases of intestinal digestion. Probiotics counts were enumeratedon MRS plates and the assay was repeated three times with duplicatesamples. Viability was also verified by performing live and deadstaining using cFDA-SE (carboxyfluorescein diacetate succinimidyl ester,50 μM) and PI (propidium iodide, 30 μM) as described (Lee et al., 2004).Levels of LAP expression in probiotic cultures during exposure to SGFand SIF were also monitored by immunofluorescence staining and Westernblotting using anti-LAP mAb. BP survival in water is also monitored toensure probiotics viability during animal feeding in a 24-h cycle.

Inhibition of L. monocytogenes adhesion, invasion and paracellulartranslocation by BP. The ability of LbcWT and BP (LbcLAP^(Lin) andLbcLAP^(Lm)) to inhibit L. monocytogenes adhesion, invasion, andtranslocation through Caco-2 cell monolayers was investigated as before(Koo et al., 2012). BLP strains were added to each well (MOE 10) andincubated for 24 h. Unbound bacteria were removed by washing withDulbecco's modified Eagles' medium containing 10% fetal calf serum(D10F), and L. monocytogenes was added (MOI 10) and incubated for 1 h todetermine inhibition of adhesion and invasion. The cell monolayers werethen washed three times and adherent bacteria were released by TRITON™X-100 treatment and plated. To determine intracellular bacteria, thecell monolayers were treated with gentamycin (50 μg/mL) for 1 h beforeTRITON™ X-100 treatment. As a vector control, the recombinant LbcVecLAP⁻strain was used.

Bacterial translocation through epithelial barrier was assayed as before(Burkholder and Bhunia, 2010). Briefly, Caco-2 cells were grown ontranswell filter inserts (4-μm pore filter; Corning, Lowell, Mass.) for10-12 days to reach confluence. Bacteria were added to the apical wellof the insert and incubated for 2 h. Liquid from the basal well wasremoved, serially diluted, and distributed onto TSA-YE agar plates forenumeration. TEER of Caco-2 cells before and after treatment wasmeasured using a Millicell ERS system (Millipore, Billerica, Mass.). Forepithelial permeability assay, 3-5 kDa FITC-Dextran (FD4; Sigma) wasadded to the well (apical side) and translocation of FD4 to the basalside was monitored by a spectrophotometer (Spectramax).

The interaction between Lactobacilli and L. monocytogenes cells. L.monocytogenes F4244, L. innocua F4248, LbcWT, LbcLAP^(Lm), andLbcLAP^(Lin) were cultured for 16-18 h at 37° C. in TSBYE, MRS, or MRSsupplemented with 2 μg/ml erythromycin broth, respectively (see section3.2.1). All cultures were pelleted by centrifugation at 8000×g for 3 minand washed with sterile PBS. All cellular concentrations were seriallydiluted to obtain a cell concentration of 10⁶ cfu/ml. L. monocytogenesor L. innocua were allowed to interact with the individual probioticstrains (LbcWT, LbcLAP^(Lm), or LbcLAP^(Lin)) at a 1:1 concentration insterile PBS for 1 h at room temperature with constant agitation on LabDoctor Revolver (MidSci, Valley Park, Mo.). Anti-Listerial magneticDynabeads (Cat. No. 71006, Thermofischer Scientific) were used tocapture and separate L. monocytogenes and L. innocua from unboundprobiotics. Briefly, 20 μl/ml of bead slurry was added to the bacterialmixtures and allowed to interact for 10 min at room temperature withconstant agitation. Beads were magnetically separated and washed withsterile PBS-T (0.1%) 3 times (10 min each wash) with constant agitation.Beads were serially diluted and plated on MOX (Neogen) and MRS agar (BD)for enumeration of Listeria and probiotics, respectively.

Mouse bioassay. Female mice (A/J: 8-10 weeks of age; n=88) werepurchased from Jackson Laboratories (Bar Harbor, Me.). The animalbioassay procedure was approved by the Purdue University Animal Care andUse Committee (1201000595). Upon arrival, mice (2/cage) were housed in acage that had a solid stainless divider to keep them separated.Shepherd's™ ALPHA-dri® (alpha cellulose) was used for bedding. Animalswere provided ad lib feed (Rodent Diet 5001, LabDiet, Brentwood, Mo.)and sterile deionized water, and acclimatized for 5 days before theexperiment. A cycle of 12 h artificial light and 12 h darkness wasmaintained. Relative humidity was 50-60% and the temperature was 20-25°C. Mice were randomly assigned to eight different groups. Freshpreparation of probiotics was supplied daily with sterile deionizedwater at ˜9×10⁹ CFU/ml for 10 days. Control animals received only water.Probiotic colonization in the gut was monitored daily by analyzing fecalcounts of probiotics on agar plates. For challenge experiment, micereceived oral gavage of L. monocytogenes F4244 (WT) at a concentrationof 5-8.8×10⁸ CFU/mouse using a feeding tube (Popper) and control micereceived PBS (Burkholder et al., 2009). Animals were observed forclinical signs, such as ruffled hair, movement and recumbency, and theirfeeding and drinking habits.

Mice were euthanized by CO₂ asphyxiation at 24 and 48 h pi, andintestine (duodenum, jejunum ileum, cecum, and colon), MLN, spleen,liver, kidney, and blood from the heart were aseptically collected.Feces were collected from each mouse from the time of infection tosacrifice. In some cases, intestinal sections were treated withgentamycin (100 μg/ml) for 2 h to kill extracellular bacteria.Organs/tissues were homogenized using a tissue homogenizer (Cole Parmer,Vernon Hills, Ill.) in 0.5 ml (blood), 4 ml (spleen, kidney, lungs) or 9ml (feces, intestine, liver) of PBS. MRS agar (Neogen, Lansing, Mich.)containing vancomycin (300 μg/ml) was used for enumeration of LbcWT, andMRS agar containing erythromycin (2 μg/ml) was used for bioengineeredstrains. Modified Oxford medium (MOX; Oxoid, Basingstoke, Hampshire, UK)was used for enumeration of Listeria. A portion of the ileum (˜2 cm) wassaved for histopathology, immunohistochemistry, qRT-PCR and otherexperiments. The gut mucosa was collected from an 8-cm section of ileumfor sIgA analysis (Haneberg et al., 1994).

Gut permeability assay. Four to five hour before sacrifice, animals wereorally gavaged with 100 μl of FD4 (3 mg/ml; Sigma). Urine voluntarilyexcreted during euthanasia, was collected from the bag, and blood wascollected by cardiac puncture. Sera and urine were appropriately dilutedand assayed for FD4 by measuring in a spectrophotometer as described(Condette et al., 2014).

Cytokine Analysis. Caco-2 monolayers (12 days of incubation) were formedin 12 well plates. Probiotics were introduced to the monolayer at an MOEof 10 and incubated for 24 h. The monolayers were challenged with L.monocytogenes (MOE 10) or LPS free purified rLAP (1 mg/ml) for 4 h(Drolia et al., 2018). Culture supernatants were collected and testedfor IL-6 and TNF-α content using ELISA kits (Raybiotech ELH-IL6 andELH-TNF-α). For mouse tissue, IL-6 and TNF-α, mouse-specific ELISA kits(Ray Biotech ELM-TNF-α and ELM-IL6-CL) were used. Briefly, ileal tissuehomogenates (100 μl) were incubated overnight (16 h). Primary antibodiesspecific to IL-6 or TNF-α and streptavidin conjugated secondaryantibodies were incubated for 1 h and 45 min, respectively, at roomtemperature. The color was developed as instructed by the manufacturer.

Histopathology and Immunohistochemistry. Mouse tissues were fixed in 4%paraformaldehyde and embedded in paraffin. Sections (5 μm thick) werestained with hematoxylin and eosin. Microscopic examination wasperformed by a board-certified veterinary pathologist and theinterpretation was based on standard histopathological morphology. Thepathologist was blinded to the treatment groups. The extent of mouseileal lesions was determined by using a semi-quantitative method thatincluded the amount of inflammatory infiltrate and percentage of gobletcells comprising the villous epithelium. A histomorphological scale forassessing inflammation in the lamina propria of the mucosa is providedas follows: 3, marked amounts (sheets of granulocytes expanding thewidth of the villous tip); 2, moderate amounts (sheets of granulocytesat the base of the villous); 1, mild amounts (multifocal scattering);and 0, none observed. To estimate percentage of goblet cells, followingscale was used: 3, 50% or greater; 2, 25-50%; 1, 11-25%; and 0, <10%.The higher the score, the more likely there is an infection in theintestinal tissues. For CD3⁺ cell staining, paraffin-embedded intestinalthin sections pre-treated with heat-induced epitope retrieval solutionand then blocked with Dako protein block according to manufacturer'sinstructions. Rabbit anti-human CD3 (1:500) used as the primary antibodyfollowed by labeling with Dako labeled polymer. The stained slides werethen scanned and analyzed using Aperio ScanScope and Aperio ImageScopesoftware (v11.2.0.780) (Aperio Technologies, Vista, Calif.) establishedalgorithms as described previously (Jones et al., 1993). For all CD3immunostained slides, a semi-quantitative histochemical score (H score)was calculated by the formula: (3×% of strongly stained)+(2×% ofmoderately stained)+(% of weakly stained), giving a range of 0 to 300.This H score was adapted from the Aperio software (Webster and Dunstan,2014).

Analysis of tight junction protein expression. Membrane proteins fromCaco-2 monolayers pre-exposed to the probiotic followed by L.monocytogenes infection were extracted and analyzed for tight junctionprotein expression. Western blot intensity measurements for membraneproteins using antibodies (Invitrogen) were determined as the ratio ofthe intensity of the tight junction protein (ZO-1, claudin-1, andoccludin) and adherens junction protein (E-cadherin, β-catenin) bands tothe integrated intensity of the β-actin band in the same sample.Additionally, membrane localization of the tight junction proteins wasalso analyzed by confocal immunofluorescence staining (Yu et al., 2012).Briefly, confluent Caco-2 monolayers were rinsed in PBS, fixed andpermeabilized in 5% formaldehyde for 15 min. The Caco-2 monolayers wereblocked using 5% normal goat serum in PBS for 1 h at room temperatureand then incubated with the primary antibody to ZO-1, Claudin-1, andOccludin or E-cadherin-1 β-catenin (Invitrogen) at 37° C. overnight. Themonolayers were then washed with PBS to remove unbound antibody and thenincubated with the FITC-conjugated secondary antibody(Anti-mouse/Anti-rabbit IgG) for 1 h at room temperature. DAPI was usedfor nuclear staining. The monolayers were then washed and imaged usingthe Leica fluorescence microscope (Leica, model DMLB, Wetzlar, Germany)equipped with SPOT software (version 4.6.4.2, Diagnostic Instruments,Sterling Heights, Mich.).

RNA preparation and quantitative reverse transcription PCR. Ileumsections (10-15 mm) of each mouse were collected and immediatelytransferred to 2.0 ml sterile, DNA/RNase-free cryovials containingRNAlater® (Ambion® by Life Technologies Corp., Carlsbad, Calif.), andstored at −80° C. until RNA extraction. Individual tissue samples werehomogenized with TRIzol® reagent (Life Technologies Corp.) using aTissue-Tearor (BioSpec Products, Inc., Bartlesville, Okla.), and totalRNA was isolated with Direct-zol™ RNA MiniPrep Plus kit (Zymo ResearchCorp., Irvine, Calif.) according to the manufacturer's instructions.Total RNA aliquots were stored at −80° C. until cDNA synthesis.Concentration and quality of the RNA samples were assessed using Agilent2100 Bioanalyzer (Agilent Technologies, Inc. Headquarters, CA).Quantitative RT-PCR was performed in two-step RT-PCR. Independent cDNAsynthesis was performed for all samples (n=3 per group) starting from100 ng of total RNA using SuperScript® VILO™ Master Mix (Invitrogen,Carlsbad, Calif.) according to the manufacturer's recommendations.Quantitative PCR was carried out in a StepOnePlus™ Real-Time PCR System(Applied Biosystems®, Foster City, Calif.) using Fast SYBR® Green MasterMix (Applied Biosystems®) according to the manufacturer's instructions.Primers for each target gene were selected from previous publications,in addition to GAPDH, chosen as an endogenous control. Three technicalreplicates for each target gene per sample were included in the qPCRassay. Means of triplicates were taken, and the relative amount oftarget mRNA was normalized to GAPDH ran in every assay. Relativequantification was evaluated using the Comparative Ct method (ΔΔCt), andfold difference (2 ^(−ΔΔct)) was calculated between control (Control)and treatment groups (Schmittgen and Livak, 2008).

Spleen cytology and flow cytometry. Mouse splenocytes (n=3 per group)were harvested by mechanical disruption through a 40-micron mesh filter(Fisher Scientific Co., Pittsburgh, Pa.) into supplemented RPMI-1640(modified Gibco, Life Technologies). Red blood cells were lysed usingACK Lysis buffer (Lonza, Allendale, N.J.). Cells were suspended in PBSwith 1% BSA prior to immunostaining. All cells were blocked withanti-mouse CD16/32 (Affymetrix, Santa Clara, Calif.). Directextracellular staining was performed. Intracellular staining for FoxP3was performed using the Mouse Regulatory T-cell Staining Kit #2(Affymetrix) according to the manufacturer's protocol. Fluorescencemeasurements were performed on an Accuri C6 flow cytometer (BD, FranklinLakes, N.J.) and analyzed with the manufacture's software. Allstatistical analyses were performed using GraphPad (GraphPad SoftwareInc, La Jolla, Calif.). Unstained and isotype control cells were usedfor preliminary gating included for all subsequent analysis. CD4⁺ andCD8α⁺ data were collected by quadrant plot (n=3). CD11c⁺ data wereobtained from detector histogram and averaged between tube 3 and tube 4for each animal (n=3). [Mean population values were compared usingtwo-way ANOVA to compare treatment groups with and without L.monocytogenes infection. Follow-up T-test analysis was performed tocompare individual treatment pairs, one-tailed tests were performed onlyif the two-tailed test showed a significant difference.]

Antibody response analysis. The gut mucosa was collected from an 8 cmsection of ileum for analysis of sIgA. Briefly, 96-well polystyreneplates (HBX, Immulon, ThermoFisher) were coated with 100 μl of mucus(diluted 1:100 in carbonate coating buffer) and stored at 4° C.overnight. The wells were washed three times in PBST and thensequentially incubated with 1:100 anti-mouse IgA conjugated to HRP andQuantaBlu substrates (Fisher). The fluorescence intensity was measured(Ex: 340 nm; Em: 420 nm) using a Spectramax fluorescent plate reader(Gemini, Sunnyvale, Calif.). Similarly, Listeria-specific IgA levelswere also estimated in the mucus samples using ELISA plates sensitizedwith an overnight culture of L. monocytogenes (Lm) F4244 (10 ⁷CFU/well), followed by exposure to mucus samples from each of the animalgroup. The presence of Lm-specific IgA was then estimated using 1:100anti-mouse IgA to HRP and QuantaBlu substrate as mentioned above. Foranalysis of serum IgG levels, 96-well plates were sensitized with serumsamples (diluted 1:100 in carbonate coating buffer) at 4° C. overnight,and the IgG levels were detected using anti-mouse IgG (1:2000) andQuantaBlu. In addition, L. monocytogenes-specific IgG response wasmeasured following sensitization with the F4244 (10 ⁷ CFU/well),followed by exposure to serum and anti-mouse IgG.

Statistical analysis. Experimental data were analyzed using MicrosoftExcel and GraphPad Prism (La Jolla, Calif.) software. For all analyses:****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, no significance. Formouse microbial counts, statistical significance was assessed by theMann-Whitney test. For the mice survival experiment, the Kaplan-Meyerplot was generated, and a log-rank test was performed. In otherexperiments, comparisons between treatment and control were performedusing the one-way or two-way analysis of variance with Tukey'smultiple-comparison test. Unless otherwise indicated, data for allexperiments are presented as the mean±standard error of the mean (SEM).

Those skilled in the art will recognize that numerous modifications canbe made to the specific implementations described above. Theimplementations should not be limited to the particular limitationsdescribed. Other implementations may be possible.

While the inventions have been illustrated and described in detail inthe drawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly certain embodiments have been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected.

It is intended that the scope of the present methods and apparatuses bedefined by the following claims. However, it must be understood thatthis disclosure may be practiced otherwise than is specificallyexplained and illustrated without departing from its spirit or scope. Itshould be understood by those skilled in the art that variousalternatives to the embodiments described herein may be employed inpracticing the claims without departing from the spirit and scope asdefined in the following claims.

REFERENCES CITED

-   Ahrne, S., and Hagslatt, M.-L. J. (2011). Effect of Lactobacilli on    paracellular permeability in the gut. Nutrients 3, 104-117.-   Amalaradjou, M. A. R., and Bhunia, A. K. (2012). Modern approaches    in probiotics research to control foodborne pathogens. Adv. Food    Nutr. Res. 67, 185-239.-   Amalaradjou, M. A. R., and Bhunia, A. K. (2013). Bioengineered    probiotics, a strategic approach to control enteric infections.    Bioengineered 4, 291-299.-   Azcarate-Peril, M. A., Sikes, M., and Bruno-Barcena, J. M. (2011).    The intestinal microbiota, gastrointestinal environment and    colorectal cancer: a putative role for probiotics in prevention of    colorectal cancer? Am J Physiol—Gastrointest Liver Physiol 301,    G401-G424.-   Bailey, T. W., do Nascimento, N. C., and Bhunia, A. K. (2017).    Genome sequence of Listeria monocytogenes strain F4244, a 4b    serotype. Genome Announcements 5, e01324-01317.-   Bakker-Zierikzee, A. M., van Tol, E. A. F., Kroes, H., Alles, M. S.,    Kok, F. J., and Bindels, J. G. (2006). Faecal SIgA secretion in    infants fed on pre- or probiotic infant formula. Ped. Allergy    Immunol. 17, 134-140.-   Bou Ghanem, E. N., Jones, G. S., Myers-Morales, T., Patil, P. D.,    Hidayatullah, A. N., and D'Orazio, S. E. (2012). InlA promotes    dissemination of Listeria monocytogenes to the mesenteric lymph    nodes during food borne infection of mice. PLoS Pathog 8, e1003015.-   Bron, P. A., Kleerebezem, M., Brummer, R.-J., Card, P. D.,    Mercenier, A., MacDonald, T. T., Garcia-Ródenas, C. L., and    Wells, J. M. (2017). Can probiotics modulate human disease by    impacting intestinal barrier function? Brit. J. Nutr. 117, 93-107.-   Brun, P., Castagliuolo, I., Leo, V. D., Buda, A., Pinzani, M., Palù,    G., and Martines, D. (2007). Increased intestinal permeability in    obese mice: new evidence in the pathogenesis of nonalcoholic    steatohepatitis. Am J. Physiol. Gastrointest. Liver Physiol. 292,    G518-G525.-   Buriti, F. C. A., Castro, I. A., and Saad, S. M. I. (2010).    Viability of Lactobacillus acidophilus in synbiotic guava mousses    and its survival under in vitro simulated gastrointestinal    conditions. Int. J. Food Microbiol. 137, 121-129.-   Burkholder, K. M., and Bhunia, A. K. (2010). Listeria monocytogenes    uses Listeria adhesion protein (LAP) to promote bacterial    transepithelial translocation, and induces expression of LAP    receptor Hsp60. Infect. Immun. 78, 5062-5073.-   Burkholder, K. M., Kim, K.-P., Mishra, K., Medina, S., Hahm, B.-K.,    Kim, H., and Bhunia, A. K. (2009). Expression of LAP, a    SecA2-dependent secretory protein, is induced under anaerobic    environment. Microbes Infect. 11, 859-867.-   Chen, W., Syldath, U., Bellmann, K., Burkart, V., and Kolb, H.    (1999). Human 60-kDa Heat-Shock Protein: A Danger Signal to the    Innate Immune System. J. Immunol. 162, 3212-3219. Cho, I.-H.,    Radadia, A. D., Farrokhzad, K., Ximenes, E., Bae, E., Singh, A. K.,    Oliver, H., Ladisch, M., Bhunia, A., Applegate, B., et al. (2014).    Nano/Micro and Spectroscopic Approaches to Food Pathogen Detection.    Annu. Rev. Anal. Chem. 7, 65-88.-   Condette, C. J., Khorsi-Cauet, H., Morliere, P., Zabijak, L.,    Reygner, J., Bach, V., and Gay-Queheillard, J. (2014). Increased gut    permeability and bacterial translocation after chronic chlorpyrifos    exposure in rats. PLoS One 9, e102217.-   Corr, S., Li, Y., Riedel, C. U., O'Toole, P. W., Hill, C., and    Gahan, C. G. M. (2007). Bacteriocin production as a mechanism for    the antiinfective activity of Lactobacillus salivarius UCC118. Proc.    Nat. Acad. Sci. (USA) 104, 7617-7621.-   Cross, M. L. (2002). Microbes versus microbes: immune signals    generated by probiotic lactobacilli and their role in protection    against microbial pathogens. FEMS Immunol. Med. Microbiol. 34,    245-253.-   Culligan, E. P., Hill, C., and Sleator, R. D. (2009). Probiotics and    gastrointestinal disease: successes, problems and future prospects.    Gut Pathog 1, 19.-   Czuprynski, C. J., Faith, N. G., and Steinberg, H. (2003). A/J mice    are susceptible and C57BL/6 mice are resistant to Listeria    monocytogenes infection by intragastric inoculation. Infect. Immun.    71, 682-689.-   Deepti, K., and Vinod, K. K. (2014). Dahi containing Lactobacillus    acidophilus and Bifidobacterium bifidum improves phagocytic    potential of macrophages in aged mice. J. Food Sci. Technol. 51,    1147-1153.-   Deshpande, G., Rao, S., Keil, A., and Patole, S. (2011).    Evidence-based guidelines for use of probiotics in preterm neonates.    BMC Med 9, 92.-   Disson, O., Grayo, S., Huillet, E., Nikitas, G., Langa-Vives, F.,    Dussurget, O., Ragon, M., Le Monnier, A., Babinet, C., Cossart, P.,    et al. (2008). Conjugated action of two species-specific invasion    proteins for fetoplacental listeriosis. Nature 455, 1114-1118.-   Drolia, R., Tenguria, S., Durkes, A. C., Turner, J. R., and    Bhunia, A. K. (2018). Listeria adhesion protein induces intestinal    epithelial barrier dysfunction for bacterial translocation. Cell    Host & Microbe 23, 470-484.-   Edelson, B. T., Bradstreet, T. R., Hildner, K., Carrero, J. A.,    Frederick, K. E., Wumesh, K. C., Belizaire, R., Aoshi, T.,    Schreiber, R. D., Miller, M. J., et al. (2011). CD8 alpha(+)    dendritic cells are an obligate cellular entry point for productive    infection by Listeria monocytogenes. Immunity 35, 236-248.-   Finlay, B. B., and Falkow, S. (1997). Common themes in microbial    pathogenicity revisited. Microbiol. Mol. Biol. Rev. 61, 136-169.-   Focareta, A., Paton, J. C., Morona, R., Cook, J., and Paton, A. W.    (2006). A recombinant probiotic for treatment and prevention of    cholera. Gastroenterology 130, 1688.-   Haneberg, B., Kendall, D., Amerongen, H. M., Apter, F. M.,    Kraehenbuhl, J. P., and Neutra, M. R. (1994). Induction of specific    immunoglobulin A in the small intestine, colon-rectum, and vagina    measured by a new method for collection of secretions from local    mucosal surfaces. Infect. Immun. 62, 15-23.-   Henderson, B., Fares, M. A., and Lund, P. A. (2013). Chaperonin 60:    a paradoxical, evolutionarily conserved protein family with multiple    moonlighting functions. Biol. Rev. 88, 955-987.-   Hill, C., Guarner, F., Reid, G., Gibson, G. R., Merenstein, D. J.,    Pot, B., Morelli, L., Canani, R. B., Flint, H. J., Salminen, S., et    al. (2014). Expert consensus document: The International Scientific    Association for Probiotics and Prebiotics consensus statement on the    scope and appropriate use of the term probiotic. Nat. Rev.    Gastroenterol. Hepatol. 11, 506-514.-   Huleatt, J. W., Pilip, I., Kerksiek, K., and Pamer, E. G. (2001).    Intestinal and splenic T cell responses to enteric Listeria    monocytogenes infection: Distinct repertoires of responding CD8 T    lymphocytes. J. Immunol. 166, 4065-4073.-   Jagadeesan, B., Fleishman Littlejohn, A. E., Amalaradjou, M. A. R.,    Singh, A. K., Mishra, K. K., La, D., Kihara, D., and Bhunia, A. K.    (2011). N-Terminal Gly₂₂₄-Gly₄₁₁ domain in Listeria adhesion protein    interacts with host receptor Hsp60. PLoS One 6, e20694.-   Jagadeesan, B., Koo, O. K., Kim, K. P., Burkholder, K. M.,    Mishra, K. K., Aroonnual, A., and Bhunia, A. K. (2010). LAP, an    alcohol acetaldehyde dehydrogenase enzyme in Listeria promotes    bacterial adhesion to enterocyte-like Caco-2 cells only in    pathogenic species. Microbiology 156, 2782-2795.-   Jagannath, C., Hoffmann, H., Sepulveda, E., Actor, J., Wetsel, R.,    and Hunter, R. (2000). Hypersusceptibility of A/J mice to    tuberculosis is in part due to a deficiency of the fifth complement    component (C5). 52, 369-379.-   Jones, M., Cordell, J. L., Beyers, A. D., Tse, A. G. D., and    Mason, D. Y. (1993). Detection of T-cell and B-cell in many animal    species using cross-reactive antipeptide antibodies. J. Immunol.    150, 5429-5435.-   Kim, H., and Bhunia, A. K. (2013). Secreted Listeria adhesion    protein (Lap) influences Lap-mediated Listeria monocytogenes    paracellular translocation through epithelial barrier. Gut Pathog.    5, 16.-   Koo, O. K., Amalaradjou, M. A. R., and Bhunia, A. K. (2012).    Recombinant probiotic expressing Listeria adhesion protein    attenuates Listeria monocytogenes virulence in vitro. PLoS One 7,    e29277.-   Lecuit, M., Dramsi, S., Gottardi, C., Fedor-Chaiken, M., Gumbiner,    B., and Cossart, P. (1999). A single amino acid in E-cadherin    responsible for host specificity towards the human pathogen Listeria    monocytogenes. EMBO J. 18, 3956-3963.-   Lecuit, M., Vandormael-Pournin, S., Lefort, J., Huerre, M., Gounon,    P., Dupuy, C., Babinet, C., and Cossart, P. (2001). A transgenic    model for listeriosis: role of internalin in crossing the intestinal    barrier. Science 292, 1722-1725.-   Lee, M. T., Chen, F. Y., and Huang, H. W. (2004). Energetics of pore    formation induced by membrane active peptides. Biochemistry 43,    3590-3599.-   Ly, N. P., Litonjua, A., Gold, D. R., and Celedon, J. C. (2011). Gut    microbiota, probiotics, and vitamin D: Interrelated exposures    influencing allergy, asthma, and obesity? J. Allergy Clin. Immunol.    127, 1087-1094.-   Ma, T. Y., Iwamoto, G. K., Hoa, N. T., Akotia, V., Pedram, A.,    Boivin, M. A., and Said, H. M. (2004). TNF-α induced increase in    intestinal epithelial tight junction permeability requires NF-kB    activation. Am. J. Physiol. Gastrointes. Liver Physiol. 286,    G367-G376.-   Maassen, C. B., Laman, J. D., den Bak-Glashouwer, M. J., Tielen, F.    J., van Holten-Neelen, J. C., Hoogteijling, L., Antonissen, C.,    Leer, R. J., Pouwels, P. H., Boersma, W. J., et al. (1999).    Instruments for oral disease-intervention strategies: recombinant    Lactobacillus casei expressing tetanus toxin fragment C for    vaccination or myelin proteins for oral tolerance induction in    multiple sclerosis. Vaccine 17, 2117-2128.-   Mantis, N. J., Rol, N., and Corthesy, B. (2011). Secretory IgA's    complex roles in immunity and mucosal homeostasis in the gut.    Mucosal Immunol. 4, 603-611.-   Marco, A. J., Altimira, J., Prats, N., Lopez, S., Dominguez, L.,    Domingo, M., and Briones, V. (1997). Penetration of Listeria    monocytogenes in mice infected by the oral route. Microb. Pathog.    23, 255-263.-   Michon, C., Langella, P., Eijsink, V. G. H., Mathiesen, G., and    Chatel, J. M. (2016). Display of recombinant proteins at the surface    of lactic acid bacteria: strategies and applications. Microb. Cell    Factories 15, 70.-   Mishra, K. K., Mendonca, M., Aroonnual, A., Burkholder, K. M., and    Bhunia, A. K. (2011). Genetic organization and molecular    characterization of secA2 locus in Listeria species. Gene 489,    76-85.-   Mohamadzadeh, M., Durmaz, E., Zadeh, M., Pakanati, K. C.,    Gramarossa, M., Cohran, V., and Klaenhammer, T. R. (2010). Targeted    expression of anthrax protective antigen by Lactobacillus gasseri as    an anthrax vaccine. Future Microbiol. 5, 1289-1296.-   Ng, S. C., Hart, A. L., Kamm, M. A., Stagg, A. J., and Knight, S. C.    (2009). Mechanisms of action of probiotics: Recent advances.    Inflamm. Bowel Dis. 15, 300-310.-   Niers, L. E. M., Timmerman, H. M., Rijkers, G. T., van Bleek, G. M.,    van Uden, N. O. P., Knol, E. F., Kapsenberg, M. L., Kimpen, J. L.    L., and Hoekstra, M. O. (2005). Identification of strong    interleukin-10 inducing lactic acid bacteria which down-regulate T    helper type 2 cytokines. Clin. Exp. Allergy 35, 1481-1489.-   Nikitas, G., Deschamps, C., Disson, O., Niault, T., Cossart, P., and    Lecuit, M. (2011). Transcytosis of Listeria monocytogenes across the    intestinal barrier upon specific targeting of goblet cell accessible    E-cadherin. J. Exp. Med. 208, 2263-2277.-   Pagnini, C., Saeed, R., Bamias, G., Arseneau, K. O., Pizarro, T. T.,    and Cominelli, F. (2010). Probiotics promote gut health through    stimulation of epithelial innate immunity. Proc. Natl. Acad. Sci.    U.S.A 107, 454-459.-   Pentecost, M., Otto, G., Theriot, J. A., and Amieva, M. R. (2006).    Listeria monocytogenes invades the epithelial junctions at sites of    cell extrusion. PLoS Pathog 2, e3.-   Pockley, A. G. (2003). Heat shock proteins as regulators of the    immune response. The Lancet 362, 469-476.-   Pouwels, P. H., Vriesema, A., Martinez, B., Tielen, F. J.,    Seegers, J. F., Leer, R. J., Jore, J., and Smit, E. (2001).    Lactobacilli as vehicles for targeting antigens to mucosal tissues    by surface exposition of foreign antigens. Methods Enzymol 336,    369-389.-   Pron, B., Boumaila, C., Jaubert, F., Sarnacki, S., Monnet, J.,    Berche, P., and Gaillard, J. (1998). Comprehensive study of the    intestinal stage of listeriosis in a rat ligated ileal loop system.    Infect. Immun. 66, 747-755.-   Rothe, J., Lesslauer, W., Lotscher, H., Lang, Y., Koebel, P.,    Kontgen, F., Althage, A., Zinkernagel, R., Steinmetz, M., and    Bluethmann, H. (1993). Mice lacking the tumour necrosis factor    receptor 1 are resistant to TNF-mediated toxicity but highly    susceptible to infection by Listeria monocytogenes. Nature 364,    798-802.-   Sakai, F., Hosoya, T., Ono-Ohmachi, A., Ukibe, K., Ogawa, A.,    Moriya, T., Kadooka, Y., Shiozaki, T., Nakagawa, H., Nakayama, Y.,    et al. (2014). Lactobacillus gasseri SBT2055 Induces TGF-beta    Expression in Dendritic Cells and Activates TLR2 Signal to Produce    IgA in the Small Intestine. PLoS One 9, e105370.-   Salminen, S., Nybom, S., Meriluoto, J., Collado, M. C., Vesterlund,    S., and El-Nezami, H. (2010). Interaction of probiotics and    pathogens—benefits to human health? Curr. Opin. Biotechnol. 21,    157-167.-   Sanders, M. E., Lenoir-Wijnkoop, I., Salminen, S., Merenstein, D.    J., Gibson, G. R., Petschow, B. W., Nieuwdorp, M., Tancredi, D. J.,    Cifelli, C. J., Jacques, P., et al. (2014). Probiotics and    prebiotics: prospects for public health and nutritional    recommendations. Annals New York Acad. Sci. 1309, 19-29.-   Schmittgen, T. D., and Livak, K. J. (2008). Analyzing real-time PCR    data by the comparative C-T method. Nat. Protoc. 3, 1101-1108.-   Schuchat, A., Swaminathan, B., and Broome, C. V. (1991).    Epidemiology of human listeriosis. Clin. Microbiol. Rev. 4, 169-183.-   Sleator, R. D., Watson, D., Hill, C., and Gahan, C. G. M. (2009).    The interaction between Listeria monocytogenes and the host    gastrointestinal tract. Microbiology 155, 2463-2475.-   Vance, R. E., Isberg, R. R., and Portnoy, D. A. (2009). Patterns of    pathogenesis: discrimination of pathogenic and nonpathogenic    microbes by the innate immune system. Cell Host Microbe 6, 10-21.-   Villena, J., Racedo, S., Aguero, G., Bru, E., Medina, M., and    Alvarez, S. (2005). Lactobacillus casei improves resistance to    pneumococcal respiratory infection in malnourished mice. J. Nutr.    135, 1462-1469.-   Wampler, J. L., Kim, K. P., Jaradat, Z., and Bhunia, A. K. (2004).    Heat shock protein 60 acts as a receptor for the Listeria adhesion    protein in Caco-2 cells. Infect. Immun. 72, 931-936.-   Webster, J. D., and Dunstan, R. W. (2014). Whole-Slide Imaging and    Automated Image Analysis: Considerations and Opportunities in the    Practice of Pathology. Vet. Pathol. 51, 211-223.-   Wollert, T., Pasche, B., Rochon, M., Deppenmeier, S., van den    Heuvel, J., Gruber, A. D., Heinz, D. W., Lengeling, A., and    Schubert, W. D. (2007). Extending the host range of Listeria    monocytogenes by rational protein design. Cell 129, 891-902.-   Xayarath, B., and Freitag, N. E. (2012). Optimizing the balance    between host and environmental survival skills: lessons learned from    Listeria monocytogenes. Future Microbiol. 7, 839-852.-   Yu, Q., Wang, Z., and Yang, Q. (2012). Lactobacillus amylophilus D14    protects tight junction from enteropathogenic bacteria damage in    Caco-2 cells. 95, 5580-5587.-   Zareie, M., Johnson-Henry, K., Jury, J., Yang, P. C., Ngan, B. Y.,    McKay, D. M., Soderholm, J. D., Perdue, M. H., and Sherman, P. M.    (2006). Probiotics prevent bacterial translocation and improve    intestinal barrier function in rats following chronic psychological    stress. Gut 55, 1553-1560.-   Zhou, Y., Qin, H., Zhang, M., Shen, T., Chen, H., Ma, Y., Chu, Z.,    Zhang, P., and Liu, Z. (2010). Lactobacillus plantarum inhibits    intestinal epithelial barrier dysfunction induced by unconjugated    bilirubin. Brit. J. Nutr. 104, 390-401.

What is claimed is:
 1. A method for treating or preventing aninflammatory condition of a patient comprising the step of administeringa therapeutically effective amount of Next Generation BioengineeredProbiotics (NGBP), together with one or more pharmaceutically acceptablecarriers, diluents, and excipients, to the patient in need of relieffrom said inflammatory condition, wherein said NGBP is a bioengineeredLactobacillus casei (L. casei) Probiotic (BLP) overexpressing apolypeptide having the sequence of SEQ ID NO:1 or SEQ ID NO:
 2. 2. Themethod of claim 1, wherein said inflammatory condition comprises Crohn'sdisease (CD), inflammatory Bowel Disease (IBD), and ulcerative colitis(UC).
 3. The method of claim 1, wherein said NGBP are a lyophilizedmaterial.
 4. The method of claim 1, wherein said NGBP are administeredorally.
 5. The method of claim 1, wherein said patient is a human beingor an animal.
 6. The method of claim 5, wherein said animal is an animalraised for food.
 7. The method of claim 5, wherein said animal is a petanimal.
 8. The method of claim 1, wherein said NGBP is added to or mixedwith the feed of said animal.
 9. The method of claim 1, wherein saidNGBP is added to or mixed with the drink of said animal.
 10. An animalfeed supplement for improving health and/or meat production of an animalcompromising Next Generation Bioengineered Probiotics (NGBP), whereinsaid NGBP comprises a bioengineered bacteria Lactobacillus casei (L.casei) expressing Listeria adhesion protein (LAP) having the sequence ofSEQ ID NO:1 or SEQ ID NO:2.
 11. The animal feed supplement of claim 10,wherein said animal is selected from the group consisting of pig, sheep,goat, chicken, turkey, cat, dog, and cattle.
 12. The animal feedsupplement of claim 10, wherein said animal feed supplement islyophilized.
 13. The animal feed supplement of claim 10, wherein saidanimal feed supplement is added to or mixed together with the feed ofsaid animal.
 14. The animal feed supplement of claim 10, wherein saidanimal feed supplement is added to or mixed together with the drink ofsaid animal.
 15. An animal feed product comprising the animal feedsupplement of claim 10.